Rationally Designed ACE2-Derived Peptides Inhibit SARS-CoV-2
- Ross C. Larue*Ross C. Larue*Email: [email protected]Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Ross C. Larue,
- Enming XingEnming XingDivision of Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Enming Xing,
- Adam D. KenneyAdam D. KenneyDepartment of Microbial Infection & Immunity, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Adam D. Kenney,
- Yuexiu ZhangYuexiu ZhangDepartment of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Yuexiu Zhang,
- Jasmine A. TuazonJasmine A. TuazonMedical Scientist Training Program, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Jasmine A. Tuazon,
- Jianrong LiJianrong LiDepartment of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Jianrong Li,
- Jacob S. YountJacob S. YountDepartment of Microbial Infection & Immunity, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Jacob S. Yount,
- Pui-Kai LiPui-Kai LiDivision of Medicinal Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Pui-Kai Li, and
- Amit Sharma*Amit Sharma*Email: [email protected]Department of Microbial Infection & Immunity, The Ohio State University, Columbus, Ohio 43210, United StatesDepartment of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Amit Sharma
Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is a novel and highly pathogenic coronavirus and is the causative agent of the coronavirus disease 2019 (COVID-19). The high morbidity and mortality associated with COVID-19 and the lack of an approved drug or vaccine for SARS-CoV-2 underscores the urgent need for developing effective antiviral therapies. Therapeutics that target essential viral proteins are effective at controlling virus replication and spread. Coronavirus Spike glycoproteins mediate viral entry and fusion with the host cell, and thus are essential for viral replication. To enter host cells, the Spike proteins of SARS-CoV-2 and related coronavirus, SARS-CoV, bind the host angiotensin-converting enzyme 2 (ACE2) receptor through their receptor binding domains (RBDs). Here, we rationally designed a panel of ACE2-derived peptides based on the RBD-ACE2 binding interfaces of SARS-CoV-2 and SARS-CoV. Using SARS-CoV-2 and SARS-CoV Spike-pseudotyped viruses, we found that a subset of peptides inhibits Spike-mediated infection with IC50 values in the low millimolar range. We identified two peptides that bound Spike RBD in affinity precipitation assays and inhibited infection with genuine SARS-CoV-2. Moreover, these peptides inhibited the replication of a common cold causing coronavirus, which also uses ACE2 as its entry receptor. Results from the infection experiments and modeling of the peptides with Spike RBD identified a 6-amino-acid (Glu37-Gln42) ACE2 motif that is important for SARS-CoV-2 inhibition. Our work demonstrates the feasibility of inhibiting SARS-CoV-2 with peptide-based inhibitors. These findings will allow for the successful development of engineered peptides and peptidomimetic-based compounds for the treatment of COVID-19.
Note
This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
Introduction
ARTICLE SECTIONS
Coronavirus disease 2019 (COVID-19) is an ongoing pandemic that has posed a serious threat to public health and the global economy. The causative agent of COVID-19 is a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV)-2, which first emerged in late 2019 in Wuhan City, China.(1,2) By March 2020, the World Health Organization had declared COVID-19 a pandemic. The rapid spread of SARS-CoV-2 is attributable to its high reproductive number, community and asymptomatic spread through close contact, and airborne transmission of respiratory droplets and aerosols.(3−5) COVID-19 patients can become critically ill with severe hypoxemia, viral pneumonia, acute respiratory distress syndrome, and gastrointestinal and neurological symptoms.(6−9) To date, there is no approved drug or vaccine for SARS-CoV-2, with the best treatments being supportive care and repurposed drugs.(10,11) A wide array of diverse approaches are urgently needed to rapidly and effectively advance antiviral therapies.
Of the seven coronaviruses known to infect humans, SARS-CoV-2 and two other highly pathogenic coronaviruses, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), are the result of zoonotic transmission.(12) Sequence analyses of coronaviruses have revealed that the SARS-CoV-2 genome shares ∼80% identity with SARS-CoV and ∼96% identity with bat coronavirus RaTG13.(1,13) Similar to all coronaviruses, SARS-CoV-2 virions display the characteristic club-shaped projections formed by trimers of viral Spike glycoprotein on their surface.(14) Spike proteins are essential for viral replication as they mediate viral entry into the host cell. During virion morphogenesis, the trimeric Spike protein is cleaved into the S1 and S2 subunits.(15−18) The cleavage event positions the receptor-binding domain (RBD) in the S1 subunits in a receptor-accessible conformation and induces structural changes in the S2 subunits to stabilize its prefusion state.(19−21) SARS-CoV and SARS-CoV-2 RBDs bind to the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), which serves as the viral entry receptor.(18,22) Binding of the RBD to ACE2 triggers another cleavage event of the S2 subunit, which results in formation of the six-helix bundle fusion core necessary for viral–host membrane fusion.(18,23−25) The essential role of Spike protein in receptor binding and viral fusion makes it a prime target for vaccine candidate development and therapeutic interventions.
Seminal SARS-CoV and SARS-CoV-2 structural studies have revealed the overall structures of Spike trimers, detailed atomic level structures of RBD bound to ACE2, and structural intermediates of the Spike-ACE2 interaction events.(19,21,26−31) These structural studies have identified that SARS-CoV and SARS-CoV-2 Spikes bind ACE2 with a nearly identical binding mode—the N-terminal lobe of the ACE2 peptidase domain binds a concave groove on the Spike RBD. Moreover, these studies highlighted that SARS-CoV and SARS-CoV-2 have conserved interactions at the RBD-ACE2 binding interface. For example, 17/20 contacting amino acid residues in ACE2 have conserved interactions with the two RBDs. Likewise, 13/14 contacting residues in the two RBDs are either conserved or have conservatively substituted side chains. Given the significance of the RBD-ACE2 interaction interface for SARS-CoV-2 infection, computational approaches have identified potential inhibitory peptides that could interfere with the interaction of Spike protein with ACE2.(32−35) These theoretical studies have suggested that regions in Spike and select residues in ACE2 could be exploited for competitive inhibition. While potentially promising, the antiviral potential of such peptides has not been experimentally evaluated.
Here, we performed comparative analyses of the SARS-CoV and SARS-CoV-2 RBD-ACE2 interaction interfaces to rationally design a panel of Spike-targeting ACE2-derived peptides (SAPs). A combination of approaches were used to evaluate the inhibitory potential, selective inhibition, and binding affinity of SAPs. Antiviral potential of selected SAPs was validated against two pathogenic human coronaviruses, SARS-CoV-2 and HCoV-NL63, both of which use ACE2 as entry receptors. Importantly, our findings provide a proof-of-principle and demonstrate feasibility of inhibiting SARS-CoV-2 infection by disrupting the Spike-ACE2 interaction interface with peptide-based inhibitors.
Results and Discussion
ARTICLE SECTIONS
Rational Design of ACE2-Derived Peptides
In order to design a panel of small peptide-based inhibitors that can block the interaction of SARS-CoV-2 Spike with the ACE2 receptor, we utilized a combination of existing structural and biochemical data, and known amino acid interactions necessary for binding of SARS-CoV and SARS-CoV-2 Spike proteins to ACE2. This included (1) crystal structures of ACE2 bound to SARS-CoV and SARS-CoV-2 Spike receptor binding domains (RBDs);(19,30,31) (2) cryoEM structures of ACE2 in complex with trimeric SARS-CoV Spike and RBD or S1 subunit of SARS-CoV-2 Spike;(21,26,29) and (3) biochemical binding data of the ACE2-interacting motif with the SARS-CoV and SARS-CoV-2 Spikes.(27,30) In particular, we focused on the Spike-ACE2 interaction interface, as it offers a prime target for competitive inhibition of viral entry. Structural and biochemical analyses have shown that SARS-CoV and SARS-CoV-2 RBDs bind ACE2 with nearly identical binding modes and with similar low nanomolar binding affinities. The α1 helix of ACE2, which is cradled in a concave groove formed by the β5 and β6 sheets of the RBD, provides the most contacts with the two RBDs (ACE2 residues Gln24, Thr27, Phe28, Lys31, His34, Glu37, Asp38, Tyr41, and Gln42). Additional contacts from ACE2 are provided by α3 helix (ACE2 residues Leu79, Met82, Tyr83 for the two RBDs), a short loop between α10 and α11 helices (ACE2 residues Gln325 and Glu329 for SARS-CoV RBD and Asn330 for both RBDs), β-hairpin flanking α11 helix (ACE2 residue Lys353 for both RBDs), and α11 helix (ACE2 residues Gly354, Asp355, and Arg357 for both RBDs). Conversely, 14 residues (402, 426, 436, 440, 442, 472, 473, 475, 479, 484, 486, 487, 488, and 491) in the two RBDs provide contacts with ACE2. These RBD residues form a network of hydrogen bonds, salt bridges, and van der Waals contacts with ACE2 residues. Based on these insights, we designed six Spike-targeting ACE2-derived peptides (SAPs)—four derived from α1, one derived from α3, and one derived from α11 helix of ACE2 (Table 1). The SAPs were designed using the following criteria: (1) they contain at least three residues predicted to interact with RBDs based on structural data (Table 1, highlighted in bold); (2) they are not highly disordered or unresolved in the crystal structures (such as ACE2 residues 1–18); and (3) the length is more than 6 and less than 30 amino acids, making them amenable for synthesis.
Table 1. Spike-Targeting ACE2-Derived Peptides (SAPs) Used in This Studya
a
Amino acid sequence with the residue number of the first and last amino acid in the sequence is indicated. The essential EDLFYQ motif is indicated in red. The SARS-CoV and SARS-CoV-2 contacting residues are indicated in bold.
SAPs Inhibit SARS-CoV-2 Spike-Pseudotyped Lentivirus Infection
We evaluated the antiviral potencies of SAPs against lentiviral vectors pseudotyped with SARS-CoV-2 Spike glycoprotein. Lentiviral cores pseudotyped with viral surface glycoproteins offer an alternative to highly pathogenic viruses that require biosafety level 3 (BSL3) or BSL4 facilities.(36) Importantly, pseudotyped viruses can be utilized at BSL2 and are ideal for studies pertaining to viral entry and screening of therapeutic agents that target viral entry, such as the peptides in this study. Luciferase-encoding lentiviruses pseudotyped with SARS-CoV-2 Spike were incubated with test peptides to allow binding to the vector particle-associated Spike prior to infection of HEK293T-ACE2 cells. Luciferase production was measured 48 h post-infection. A titration curve for each peptide was generated for determining its inhibitory concentration (IC50). Of the six SAPs tested, SAP1, SAP2, and SAP6 inhibited SARS-CoV-2 Spike-mediated virus infection with an IC50 value of 2.39 ± 0.20, 3.72 ± 0.37, and 1.90 ± 0.14 mM, respectively (Figure 1). In contrast, 50% inhibition of Spike-mediated virus infection was not achieved with SAP3, SAP4, or SAP5 even at 7.5 mM, the highest concentration tested. Thus, three of the six SAPs inhibit SARS-CoV-2 Spike-mediated virus infection with IC50 values in the low millimolar range.
Figure 1
Figure 1. Dose-dependent inhibition of SARS-CoV-2 Spike-pseudotyped lentivirus infection by SAPs. Dose response curves of the indicated SAPs generated by plotting the percent viral inhibition (y-axis) against the log transformation of SAP concentration (mM, x-axis). Each data point represents the average of three independent experiments, performed in duplicate. Error bars represent standard deviations. The dotted gray line indicates 50% viral inhibition used to determine the IC50 value. Computed IC50 values for the indicated SAPs from three independent experiments ± standard deviations are shown.
Despite the fact that the genomes of SARS-CoV and SARS-CoV-2 share ∼80% sequence identity and most of the sequence variation is within the Spike open reading frame,(1) the overall structure and ACE2-binding mode of their Spike RBDs are nearly identical. Moreover, the majority of amino acid residues in the SARS-CoV and SARS-CoV-2 RBDs that are essential for binding ACE2 either are identical or have conserved side chains.(29−31) Thus, we sought to determine whether SAPs that inhibit SARS-CoV-2 Spike-mediated virus infection are also able to inhibit infection mediated by SARS-CoV Spike. For this, SAPs with positive inhibitory profiles from Figure 1 were evaluated for their ability to inhibit infection of SARS-CoV Spike- and SARS-CoV-2 Spike-pseudotyped viruses at 3 mM dose, which is within the IC50 range for the test peptides (Figure 1). As specificity control, antiviral activity of SAPs was also measured against lentiviruses pseudotyped with vesicular stomatitis virus Glycoprotein (VSV-G), which utilizes low-density lipoprotein receptor, LDL-R, for viral entry.(37) In comparison to the diluent control, SAP1, SAP2, and SAP6 treatment resulted in ∼1.6–3.5-fold reduction in SARS-CoV-2 Spike-mediated infection (Figure 2A) and ∼1.9–7.5-fold reduction in SARS-CoV Spike-mediated infection (Figure 2B). Consistent with the results in Figure 1, SAP5 treatment had no significant effect on SARS-CoV-2 Spike-mediated infection, but resulted in ∼1.5-fold reduction in SARS-CoV Spike-mediated infection. None of the SAPs affected VSV-G-mediated virus infection demonstrating their specificity for inhibiting Spike-mediated viral entry (Figure 2C). The slightly higher potency of SAP1, SAP5, and SAP6 against SARS-CoV Spike-mediated infection compared to SARS-CoV-2 Spike-mediated infection could be attributable to subtle differences in the SARS-CoV and SARS-CoV-2 RBD-ACE2 interaction interfaces.(29−31) Importantly, these results highlight the fact that minor differences in the number of contact residues and their interactions at the RBD-ACE2 interface could be exploitable for structure-based rational design of viral-specific inhibitors.
Figure 2
Figure 2. Inhibition of Spike- and VSV-G-pseudotyped lentivirus infection by SAPs. Luciferase-encoding lentiviruses pseudotyped with indicated viral glycoprotein were incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of 293T-ACE2 cells. Infection was measured as relative luciferase expression 48 h post-infection. The luciferase signal obtained for the diluent control was set to 100%. Graphs indicate the percentage of infected cells normalized to the diluent control for lentiviruses pseudotyped with (A) SARS-CoV-2 Spike, (B) SARS-CoV Spike, or (C) VSV-G. Bars represent averages from four independent experiments, performed in duplicate, with means from individual experiments shown as circles. Error bars represent standard deviations. Percent infections were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
Binding Affinities of SAPs to SARS-CoV-2 Spike RBD
Recent studies using biochemical and biophysical methods have demonstrated that the RBD within the S1 subunit of SARS-CoV-2 Spike is responsible for binding ACE2 with high affinity.(27,30) Thus, we employed affinity precipitation assays to determine the binding affinities of SAPs with positive inhibitory profiles to recombinantly expressed and purified Spike RBD. A titration curve for each FITC-labeled peptide was generated for determining its binding affinity to His-tagged Spike RBD (Figure 3A and B). Of the four SAPs tested, SAP1 displayed the highest binding affinity (Kd = 0.53 ± 0.01 mM), whereas SAP5 did not display any detectable binding in vitro (Figure 3B). SAP6, which contains the overlapping region in SAP1 and SAP2 (Table 1), displayed binding affinity similar to that of SAP1 (Figure 3C, SAP6 Kd of 1.36 ± 0.14 mM vs SAP1 Kd = 0.53 ± 0.01 mM). SAP2 displayed lower binding (Kd = 10.7 ± 4.2 mM) possibly attributable to the presence of two consecutive serine residues in the peptide, which could affect its flexibility. Thus, our results suggest that SAP6 contains the minimal residues needed for binding RBD, and additional residues in SAP1 can slightly improve the binding affinity. Moreover, we found that the in vitro binding affinities of SAPs track closely with their antiviral IC50 values (Figures 1 and 3).
Figure 3
Figure 3. Binding of SAPs to SARS-CoV-2 Spike. Affinity precipitation of His-tagged SARS-CoV-2 Spike RBD (indicated “His-S RBD”) with FITC-SAPs. (A) Representative SDS-PAGE gels of affinity precipitation of His-S RBD with increasing concentrations of indicated FITC-SAP (lanes 2–8: 0.125, 0.25, 0.5, 1, and 3 mM FITC-SAP). Lane 1 indicates control precipitation of 3 mM FITC-SAP without His-S RBD. FITC-labeled bands were detected at 488 nm fluorescence and His-S RBD was visualized with Coomassie staining. (B) Graphical representation of fluorescence intensities from (A) of indicated FITC-SAP bound to His-S RBD. Each data point represents the average of three independent experiments. Error bars represent standard deviations. Data were fit to the Hill equation to determine the apparent Kd of binding. (C) Calculated binding Kd from three independent experiments ± standard deviations.
SAP1 and SAP6 Inhibit SARS-CoV-2 Infection
We next sought to determine whether SAPs that bind RBD with high affinity and display positive inhibitory profiles against pseudotyped viruses are also able to inhibit the infection of genuine SARS-CoV-2. To this end, SAP1, SAP5, and SAP6 were evaluated for their ability to inhibit infection of SARS-CoV-2. Viruses was incubated with 3 mM of test peptide or diluent control to allow binding to the virion-associated Spike prior to infection of HEK293T-ACE2-GFP cells. Percent infection was measured by intracellular staining of SARS-CoV-2 N protein and flow cytometry 24 h post-infection. In comparison to the diluent control, SAP1 and SAP6 treatment resulted in ∼2-fold reduction in SARS-CoV-2 infection (Figure 4A and B). In contrast, SAP5, which does not bind RBD and does not inhibit SARS-CoV-2 Spike-pseudotyped lentiviruses, had no significant effect on virus infection. Based on the findings that SAP1 and SAP6 have comparable IC50 values (Figure 1), bind RBD with similar affinity (Figure 3C), and inhibit SARS-CoV-2 infection to similar levels (Figure 4B), we conclude that SAP6 contains the minimal necessary residues for inhibition of SARS-CoV-2.
Figure 4
Figure 4. Inhibition of SARS-CoV-2 infection by SAPs. SARS-CoV-2 was incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of 293T-ACE2-GFP cells. Infection was measured by flow cytometry as the percentage of cells positive for SARS-CoV-2 nucleocapsid (N) protein 24 h post-infection. (A) Representative flow cytometry plots indicating percent infection. (B) Graph indicates the percentage of infected cells normalized to the diluent control, which was set to 100%. Bars represent averages from two independent experiments, performed in triplicate, with individual data points shown as circles. Error bars represent standard deviations. Percent infections were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
SAP1 and SAP6 Inhibit HCoV-NL63 Infection
In addition to the three highly pathogenic coronaviruses known to infect humans, four low pathogenicity coronaviruses (HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63) are endemic in humans and cause common cold and upper and lower respiratory tract infections.(38−40) Of the four endemic human coronaviruses, only HCoV-NL63 uses ACE2 as an entry receptor.(41) Similar to SARS-CoV and SARS-CoV-2, the S1 subunit of HCoV-NL63 Spike binds ACE2 to mediate viral entry.(42) Thus, we sought to determine whether SAP1 and SAP6, which inhibit SARS-CoV-2 infection, could also inhibit HCoV-NL63 infection. Viruses were incubated with 3 mM of test peptide or diluent control to allow binding to the virion-associated Spike prior to infection of LLC-MK2 cells. Virus-induced cytopathic effects (CPEs) and virus titers in the supernatants were measured 72 h post-infection. Severe CPEs were observed in cells that were infected with viruses treated with diluent control or SAP5 indicating robust viral infection (Figure 5A). In contrast, reduced CPEs were observed in cells infected with SAP1- or SAP6-treated viruses indicating reduced viral infection. Moreover, in comparison to the diluent control, SAP1 and SAP6 treatment resulted in ∼3-fold reduced HCoV-NL63 titers (Figure 5B). In contrast, SAP5 treatment did not result in significant reduction of viral titers. Taken together, our results demonstrate that SAP1 and SAP6 inhibit infection of SARS-CoV-2 and HCoV-NL63, both of which utilize ACE2 as entry receptor.
Figure 5
Figure 5. Inhibition of HCoV-NL63 infection by SAPs. HCoV-NL63 was incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of LLC-MK2 cells. Cytopathic effects and virus titers in the supernatants were analyzed at 72 h post-infection. (A) Representative bright field microscope images showing cytopathic effects. (B) Graph indicates virus titers in supernatants from LLC-MK2 cells. Bars represent averages from triplicate infections with individual data points shown as circles. Error bars represent standard deviations. Virus titers were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
Structural Modeling of SAP1 and SAP6 with SARS-CoV-2 Spike
The findings from our infectivity assays suggest that SAP1 and SAP6, and to a lesser degree SAP2, block the interaction of SARS-CoV-2 Spike with ACE2. We found that both SAP1 and SAP6 inhibit SARS-CoV-2 infection to similar levels. Since SAP6 contains the minimal conserved short EDLFYQ motif present in SAP1 and SAP2, we conclude that it is the minimal essential motif important for inhibition of SARS-CoV-2. The cocrystal structure of SARS-CoV-2 Spike RBD and human ACE2 has been recently solved and available through the Protein Data Bank (PDB).(30) As shown in Figure 6A, magenta surface and ribbon represent the Spike RBD and yellow ribbon corresponds to ACE2. The RBD-ACE2 interaction interface is contacted mainly by the N-terminal helix (residues Ile21-Asn51) of ACE2. Our results suggest that SAP6 (Glu37-Gln42, blue ribbon in Figure 6B) and SAP1 (Thr27-Gln42, blue and green ribbon in Figure 6B) are able to disrupt the RBD-ACE2 interaction in the low millimolar range, indicating the importance of these residues at the N-terminal helix of ACE2 for RBD-ACE2 interaction. Based on the crystal structure solved by Lan et al.(30) and highlighted in the modeling studies,(28) polar residues (Glu37, Asp38, Tyr41, and Gln42) of SAP6 are able to form a network of hydrogen bonds with Thr500, Tyr449, Asn501, and Tyr505 of SARS-CoV-2 Spike RBD (Figure 6C). In particular, the carboxy groups of Glu37, Asp38, and Gln42 and hydroxyl group of Tyr41 of SAP6 interface with Spike cavity surrounded by Gln498, Thr500, Tyr449, Asn501, and Tyr505. Taken together, these structural insights lend support to our identification of SAP1 and SAP6 as peptide disruptors of the Spike RBD-ACE2 interaction.
Figure 6
Figure 6. Graphical illustration of SARS-CoV-2 Spike and SAP6 interaction interface. (A) Overall view of SARS-CoV-2 Spike RBD and human ACE2 interaction mode. The N-terminal helix of human ACE2 is located at the central interface. (B) Relative location of SAP6 (light blue) and SAP1 (green and light blue). (C) H-bond interaction network between SAP6 and SARS-CoV-2 Spike RBD. The Y41, Q42, D38, and E37 of SAP6 peptide are involved in H-bond interactions with T500, Y449, N501, and Y505 of SARS-CoV-2 Spike RBD. Corresponding crystal structure: PDB Code: 6M0J. http://www.rcsb.org/structure/6M0J.
Conclusions
ARTICLE SECTIONS
In summary, we have developed and screened a panel of rationally designed, small peptide inhibitors and identified peptides that block the interaction of coronavirus Spike proteins with ACE2. Importantly, we have identified two peptides, SAP1 and SAP6, which inhibit SARS-CoV-2 infection—demonstrating the feasibility of targeting Spike–ACE2 interaction interface with peptide-based inhibitors to inhibit virus infection. SAP6, which contains the minimal conserved EDLFYQ sequence, highlights the importance of the N-terminal α1 helix of ACE2 for interaction with Spike protein. Future structure-based rational design studies focused on improved conformational matching between SAPs and SARS-CoV-2 Spike protein will allow for increased binding affinity and potent viral inhibition. Such approaches could include increased noncovalent π–π interactions between aromatic amino acid residues and the enhancement of peptide α-helicity to increase the stability of the SAPs. Lending support to such approaches, a recent study employed computer-generated scaffolds built around the α1 helix of ACE2 to design de novo miniprotein inhibitors of SARS-CoV-2.(43) In summary, our proof-of-principle study that SARS-CoV-2 can be inhibited by small peptides will further allow for the successful development of engineered peptides and peptidomimetic-based compounds for the treatment of COVID-19.
Methods
ARTICLE SECTIONS
Peptide Design and Recombinant Proteins
SAPs were designed using the following published structures: SARS-CoV-2 Spike S1 subunit bound to ACE2 (PDB codes: 7A91–98), SARS-CoV-2 RBD bound to ACE2 (PDB codes: 6M0J and 6VW1), SARS-CoV RBD bound to ACE2 (PDB code: 2AJF), and SARS-CoV S1–S2 subunits bound to ACE2 (PDB codes: 6ACK, 6ACJ, 6ACC, 6ACD, and 6ACG).(19,21,26−31) Sequence and structural comparisons of the SARS-CoV and SARS-CoV-2 binding interfaces with ACE2 were performed using Clustal Omega (EMBL-EBI, England), SWISS-MODEL (Biozentrum, Switzerland), and MUSTER (University of Michigan, USA). Once designed, synthetic SAPs were purchased from Biomatik (95% purity, with TFA removed) either unmodified or with an N-terminal FITC label (FITC-SAP). 1× Phosphate Buffered Saline (PBS) was used as a diluent to reconstitute SAPs. To improve the solubility of SAP1 and SAP2, 1× PBS was supplemented with 10% and 5% aqueous NH3, respectively. Recombinant His-tagged SARS-CoV-2 Spike RBD (His-S RBD, amino acids Arg319-Phe541) was purchased from RayBiotech.
Affinity Precipitation Assay
Affinity precipitation assays using Ni-NTA beads (GE Healthcare) were performed with His-S RBD and FITC-SAPs using previously described methods.(44) Ni-NTA beads were equilibrated in binding buffer (50 mM Tris pH 7.5, 250 mM NaCl, 50 mM imidazole, and 2 mM β-mercaptoethanol). Binding reactions were setup by incubating equilibrated Ni-NTA beads with His-S RBD (1 μM) and increasing concentrations (3, 1, 0.5, 0.25, and 0.125 mM) of indicated FITC-SAP in the binding buffer and incubated for 1 h at 4 °C. In parallel, control reactions with 3 mM of indicated FITC-SAP without His-S RBD were preformed to rule out nonspecific FITC-SAP binding to the Ni-NTA beads. Reactions were spun and washed three times in the binding buffer to remove unbound proteins/peptides. The resulting protein–peptide complexes bound to the beads were extracted using NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen), subjected to SDS-PAGE analysis, and visualized by Coomassie staining or fluorescence detection at 488 nm. Resulting FITC-labeled bands were quantified using ImageJ software. To estimate Kd values for FITC-SAP binding to His-S RBD, the data were fit to the Hill equation using Origin 8 software (OriginLab).
Cells, Plasmids, Viruses
HEK293T (ATCC CRL-3216), HEK293T-ACE2 (BEI Resources), Vero E6 (ATCC CRL-1586), and LLC-MK2 (ATCC CCL-7) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). HEK293T cells stably expressing GFP-tagged human ACE2 (HEK293T-ACE2-GFP) were generated using methods described previously,(45) and were maintained in DMEM supplemented with 10% FBS and 1 μg/mL puromycin (Sigma).
Plasmid encoding SARS-CoV-2 Spike (pCAGGS-SARS-CoV-2 Spike) was obtained from BEI Resources. Generation of plasmid encoding SARS-CoV Spike (pCAGGS-SARS-CoV Spike) has been described previously.(46) Vesicular stomatitis virus-G (VSV-G) expression plasmid (pMD2.G) was purchased from Addgene. HIV-1-derived luciferase reporter vector (pNL4–3.Luc.R–E–) was obtained from NIH AIDS Reagent Program.
SARS-CoV-2 USA-WA1/2020 stock virus was obtained from BEI Resources. Human coronavirus NL63 (HCoV-NL63) stock was obtained from Dr. Susan Baker (Loyola University, Chicago, IL).
Pseudovirus Production
Luciferase-encoding lentiviruses pseudotyped with viral glycoprotein of interest were generated using methods described previously.(47) Briefly, HEK293T cells were transfected with pNL4–3.Luc.R–E– and pCAGGS-SARS-CoV-2 Spike, pCAGGS-SARS-CoV Spike, or pMD2.G using Fugene 6 transfection reagent (Roche) following manufacturer’s protocol. Forty-eight hours post-transfection, virus-containing supernatants were harvested, filtered through 0.45 μm sterile filter, and concentrated using Amicon Ultra-15 centrifugal filters (Millipore). Aliquots of pseudoviruses were stored at −80 °C. The titers of SARS-CoV-2 Spike-, SARS-CoV Spike-, and VSV-G-pseudotyped viruses were in the range of ∼2 × 105, ∼1 × 105, and ∼3 × 107 relative luciferase units (RLUs)/mL, respectively.
Pseudovirus Inhibition Assay
HEK293T-ACE2 cells were seeded in μClear Black 96-well plates (Greiner Bio-One) in 100 μL of DMEM supplemented with 10% FBS at a density of 1.25 × 104 cells per well. Sixteen hours after plating, equal amounts (RLUs/mL) of a given pseudovirus were incubated with indicated concentrations of the test peptide or diluent control (1× PBS) in 50 μL of DMEM supplemented with 10% FBS for 1 h at 37 °C in a V-bottom 96-well plate. The virus–peptide mixture was then added to the HEK293T-ACE2 cells. Polybrene (Sigma) at the final concentration of 5 μg/mL was added to the cells. After 48 h, 100 μL of supernatant was removed from each well and luciferase activity was measured using Bright-Glo Luciferase Assay System (Promega) following the manufacturer’s protocol. Luminescence was detected using an Infinite M PLEX multimode plate reader (Tecan).
Dose Response Curves
For dose response curves, the pseudovirus inhibition assays were performed with increasing concentrations of SAP (0.001, 0.01, 0.1, 1.0, 2.5, 5.0, and 7.5 mM). Percent viral inhibition was calculated as the percent reduction in luciferase activity of pseudovirus incubated with a given concentration of SAP compared to the pseudovirus incubated with the diluent control. The concentration of SAP that resulted in 50% inhibition of viral replication (IC50) was interpolated from a nonlinear, best-fit curve using GraphPad Prism 8.0.2 software.
Biosafety Procedures for Live SARS-CoV-2 Experiments
All experiments involving live SARS-CoV-2 were performed at Biosafety Level 3 (BSL3) according to the standard operating procedures approved by The Ohio State University BSL3 Operations Group (BOG) and Institutional Biosafety Committee. Infected cells were removed from the BSL3 facility for subsequent analyses after fixation with 4% paraformaldehyde for a minimum of 1 h in accordance with a validated decontamination protocol approved by the BOG and Institutional Biosafety Officer.
SARS-CoV-2 Propagation
SARS-CoV-2 USA-WA1/2020 stock was diluted 1:10,000 in DMEM and added to confluent Vero E6 cells. After infection for 1 h at 37 °C, media was replaced with DMEM supplemented with 4% FBS. Following incubation at 37 °C for 72 h, virus-containing supernatant was clarified at 1000g for 10 min to remove cell debris, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. The viral stock titer was determined by plaque assay on Vero E6 cells in 12-well plates with 0.3% low-melting agarose (Sigma) overlay and visualization with 0.25% crystal violet (Sigma).
SARS-CoV-2 Infections
106 plaque-forming units of SARS-CoV-2 were incubated with 3 mM final concentration of the test peptide or diluent control (1× PBS) in 400 μL of DMEM for 1 h at 37 °C. The virus–peptide mixture was then added to near-confluent HEK293T-ACE2-GFP cells in a 12-well plate. Infection was allowed to proceed for 1 h at 37 °C at which point media was removed and replaced with 500 μL of DMEM supplemented with 10% FBS. After 24 h, the cells were harvested using 0.25% trypsin-EDTA (Gibco), fixed with 4% paraformaldehyde (Thermo Scientific) for 1 h at room temperature, permeabilized with 1× PBS containing 0.1% Triton X-100, and blocked with 1× PBS containing 2% FBS. The cells were then stained with anti-SARS-CoV-2 N mouse monoclonal antibody (1:1000, Sino Biological) followed by staining with anti-mouse AlexaFluor-647 secondary antibody (1:1000, Life Technologies). The stained cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data was analyzed using FlowJo software.
HCoV-NL63 Propagation
HCoV-NL63 stock was expanded and titered on LLC-MK2 cells as described previously.(48) The virus was aliquoted and stored at −80 °C.
HCoV-NL63 Infections
HCoV-NL63 equiv to MOI of 0.5 was incubated with 3 mM final concentration of the test peptide or diluent control (1× PBS) in 300 μL of DMEM for 1 h at 37 °C. Confluent LLC-MK2 cells in 6-well plates were washed once with DMEM. The virus–peptide mixture was then added to LLC-MK2 cells in triplicate. Infection was allowed to proceed for 1 h at 37 °C at which point media was removed and replaced with 1 mL of DMEM supplemented with 2% FBS. After 72 h, the cytopathic effects (CPE) in each well were imaged under a light microscope. The cell culture supernatants were collected for virus titration by plaque assay.
HCoV-NL63 Plaque Assay
Confluent LLC-MK2 cells in 6-well plates were infected with serial dilutions (ranging from 10–1 to 10–7) of HCoV-NL63 in DMEM. After infection for 1 h at 37 °C, cells were washed three times with DMEM and overlaid with 1% low-melting agarose in 2 mL of DMEM supplemented with 2% FBS. After incubation at 37 °C for 5 days, cells were fixed with 4% paraformaldehyde for 2 h, and plaques were visualized after staining with 0.05% crystal violet.
Author Contributions
Conceptualization: R.C.L. and A.S.; research design: R.C.L., E.X., J.L., J.S.Y., P–K.L., A.S.; investigation: R.C.L., E.X., A.D.K., Y.Z., J.A.T., J.S.Y., A.S.; writing—original draft: R.C.L. and A.S.; writing—review and editing: all authors.
The authors declare the following competing financial interest(s): Ross C Larue and Amit Sharma have filed a provisional patent application for these inhibitors.
Author Information
ARTICLE SECTIONS
- Amit Sharma - Department of Microbial Infection & Immunity, The Ohio State University, Columbus, Ohio 43210, United States; Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, United States;
http://orcid.org/0000-0002-3836-5617;
Conceptualization: R.C.L. and A.S.; research design: R.C.L., E.X., J.L., J.S.Y., P–K.L., A.S.; investigation: R.C.L., E.X., A.D.K., Y.Z., J.A.T., J.S.Y., A.S.; writing—original draft: R.C.L. and A.S.; writing—review and editing: all authors.
The authors declare the following competing financial interest(s): Ross C Larue and Amit Sharma have filed a provisional patent application for these inhibitors.
Acknowledgments
ARTICLE SECTIONS
This research was supported by The Ohio State University’s institutional start-up funds to R.C.L. and A.S. We thank Geraldine Vilmen and Natalie Cyberski for technical assistance and helpful discussions. The ToC graphic was created with BioRender.com.
References
ARTICLE SECTIONS
This article references 48 other publications.
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- 8Mao, R., Qiu, Y., He, J. S., Tan, J. Y., Li, X. H., Liang, J., Shen, J., Zhu, L. R., Chen, Y., Iacucci, M. (2020) Manifestations and prognosis of gastrointestinal and liver involvement in patients with COVID-19: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol 5, 667– 678, DOI: 10.1016/S2468-1253(20)30126-6[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38vms12msw%253D%253D&md5=1adb94e19792a53f7a3b2621cc2ace0bManifestations and prognosis of gastrointestinal and liver involvement in patients with COVID-19: a systematic review and meta-analysisMao Ren; Chen Min-Hu; Qiu Yun; He Jin-Shen; Tan Jin-Yu; Li Xue-Hua; Liang Jie; Shen Jun; Zhu Liang-Ru; Chen Yan; Iacucci Marietta; Ghosh Subrata; Ng Siew CThe lancet. Gastroenterology & hepatology (2020), 5 (7), 667-678 ISSN:.BACKGROUND: The prevalence and prognosis of digestive system involvement, including gastrointestinal symptoms and liver injury, in patients with COVID-19 remains largely unknown. We aimed to quantify the effects of COVID-19 on the digestive system. METHODS: In this systematic review and meta-analysis, we systematically searched PubMed, Embase, and Web of Science for studies published between Jan 1, 2020, and April 4, 2020. The websites of WHO, CDC, and major journals were also searched. We included studies that reported the epidemiological and clinical features of COVID-19 and the prevalence of gastrointestinal findings in infected patients, and excluded preprints, duplicate publications, reviews, editorials, single case reports, studies pertaining to other coronavirus-related illnesses, and small case series (<10 cases). Extracted data included author; date; study design; country; patient demographics; number of participants in severe and non-severe disease groups; prevalence of clinical gastrointestinal symptoms such as vomiting, nausea, diarrhoea, loss of appetite, abdominal pain, and belching; and digestive system comorbidities including liver disease and gastrointestinal diseases. Raw data from studies were pooled to determine effect estimates. FINDINGS: We analysed findings from 35 studies, including 6686 patients with COVID-19, that met inclusion criteria. 29 studies (n=6064) reported gastrointestinal symptoms in patients with COVID-19 at diagnosis, and the pooled prevalence of digestive system comorbidities was 4% (95% CI 2-5; range 0-15; I(2)=74%). The pooled prevalence of digestive symptoms was 15% (10-21; range: 2-57; I(2)=96%) with nausea or vomiting, diarrhoea, and loss of appetite being the three most common symptoms. The pooled prevalence of abnormal liver functions (12 studies, n=1267) was 19% (9-32; range 1-53; I(2)=96%). Subgroup analysis showed patients with severe COVID-19 had higher rates of abdominal pain (odds ratio [OR] 7·10 [95% CI 1·93-26·07]; p=0·003; I(2)=0%) and abnormal liver function including increased ALT (1·89 [1·30-2·76]; p=0·0009; I(2)=10%) and increased AST (3·08 [2·14-4·42]; p<0·00001; I(2)=0%) compared with those with non-severe disease. Patients in Hubei province, where the initial COVID-19 outbreak occurred, were more likely to present with abnormal liver functions (p<0·0001) compared with those outside of Hubei. Paediatric patients with COVID-19 had a similar prevalence of gastrointestinal symptoms to those of adult patients. 10% (95% CI 4-19; range 3-23; I(2)=97%) of patients presented with gastrointestinal symptoms alone without respiratory features. Patients who presented with gastrointestinal system involvement had delayed diagnosis (standardised mean difference 2·85 [95% CI 0·22-5·48]; p=0·030; I(2)=73%). Patients with gastrointestinal involvement tended to have a poorer disease course (eg, acute respiratory distress syndrome OR 2·96 [95% CI 1·17-7·48]; p=0·02; I(2)=0%). INTERPRETATION: Our study showed that digestive symptoms and liver injury are not uncommon in patients with COVID-19. Increased attention should be paid to the care of this unique group of patients. FUNDING: None.
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- 11Berlin, D. A., Gulick, R. M., and Martinez, F. J. (2020) Severe Covid-19. N. Engl. J. Med. DOI: 10.1056/NEJMcp2009575
- 12Cui, J., Li, F., and Shi, Z. L. (2019) Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17, 181– 192, DOI: 10.1038/s41579-018-0118-9[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVyhtbnL&md5=b2b2c40c0cdf30107d8c70f482fc4f63Origin and evolution of pathogenic coronavirusesCui, Jie; Li, Fang; Shi, Zheng-LiNature Reviews Microbiology (2019), 17 (3), 181-192CODEN: NRMACK; ISSN:1740-1526. (Nature Research)A review. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are two highly transmissible and pathogenic viruses that emerged in humans at the beginning of the 21st century. Both viruses likely originated in bats, and genetically diverse coronaviruses that are related to SARS-CoV and MERS-CoV were discovered in bats worldwide. In this Review, we summarize the current knowledge on the origin and evolution of these two pathogenic coronaviruses and discuss their receptor usage; we also highlight the diversity and potential of spillover of bat-borne coronaviruses, as evidenced by the recent spillover of swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs.
- 13Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., Wang, W., Song, H., Huang, B., Zhu, N. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565– 574, DOI: 10.1016/S0140-6736(20)30251-8[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOmsLY%253D&md5=3a644f602ea1c68114e23b5f12625265Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor bindingLu, Roujian; Zhao, Xiang; Li, Juan; Niu, Peihua; Yang, Bo; Wu, Honglong; Wang, Wenling; Song, Hao; Huang, Baoying; Zhu, Na; Bi, Yuhai; Ma, Xuejun; Zhan, Faxian; Wang, Liang; Hu, Tao; Zhou, Hong; Hu, Zhenhong; Zhou, Weimin; Zhao, Li; Chen, Jing; Meng, Yao; Wang, Ji; Lin, Yang; Yuan, Jianying; Xie, Zhihao; Ma, Jinmin; Liu, William J.; Wang, Dayan; Xu, Wenbo; Holmes, Edward C.; Gao, George F.; Wu, Guizhen; Chen, Weijun; Shi, Weifeng; Tan, WenjieLancet (2020), 395 (10224), 565-574CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)In late Dec., 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions detd. by rapid amplification of cDNA ends. Phylogenetic anal. of these 2019-nCoV genomes and those of other coronaviruses was used to det. the evolutionary history of the virus and help infer its likely origin. Homol. modeling was done to explore the likely receptor-binding properties of the virus. The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic anal. revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homol. modeling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues.2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic anal. suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural anal. suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University. These data have been deposited in the ChinaNational Microbiol. Data Center (accession no. NMDC10013002 and genome accession nos. NMDC60013002-01 to NMDC60013002-10) and the datafrom BGI have been deposited in the China National GeneBank (accession nos. CNA000733235).
- 14Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., Graham, B. S., and McLellan, J. S. (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260– 1263, DOI: 10.1126/science.abb2507[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFemt70%253D&md5=27d08cbb9a43d1da051a8a92a9f68aa5Cryo-EM structure of the 2019-nCoV spike in the prefusion conformationWrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
- 15Belouzard, S., Chu, V. C., and Whittaker, G. R. (2009) Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc. Natl. Acad. Sci. U. S. A. 106, 5871– 5876, DOI: 10.1073/pnas.0809524106[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkvFejtrY%253D&md5=bec80a49cc00c0ebf06a5906ea5c1ccaActivation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sitesBelouzard, Sandrine; Chu, Victor C.; Whittaker, Gary R.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (14), 5871-5876CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a crit. proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.
- 16Simmons, G., Reeves, J. D., Rennekamp, A. J., Amberg, S. M., Piefer, A. J., and Bates, P. (2004) Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc. Natl. Acad. Sci. U. S. A. 101, 4240– 4245, DOI: 10.1073/pnas.0306446101[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFartLw%253D&md5=572fe2967e6bd433308eaf39e27e1930Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entrySimmons, Graham; Reeves, Jacqueline D.; Rennekamp, Andrew J.; Amberg, Sean M.; Piefer, Andrew J.; Bates, PaulProceedings of the National Academy of Sciences of the United States of America (2004), 101 (12), 4240-4245CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addn., we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Addnl., there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.
- 17Shang, J., Wan, Y., Luo, C., Ye, G., Geng, Q., Auerbach, A., and Li, F. (2020) Cell entry mechanisms of SARS-CoV-2. Proc. Natl. Acad. Sci. U. S. A. 117, 11727– 11734, DOI: 10.1073/pnas.2003138117[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1aitL3K&md5=07c0a8dc7eca57b6878841d1441ce8aeCell entry mechanisms of SARS-CoV-2Shang, Jian; Wan, Yushun; Luo, Chuming; Ye, Gang; Geng, Qibin; Auerbach, Ashley; Li, FangProceedings of the National Academy of Sciences of the United States of America (2020), 117 (21), 11727-11734CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochem. and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.
- 18Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280, DOI: 10.1016/j.cell.2020.02.052[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktl2qtb8%253D&md5=60aea5c939a2d4df034a91d6198fb3efSARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease InhibitorHoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, StefanCell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
- 19Li, F., Li, W., Farzan, M., and Harrison, S. C. (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864– 1868, DOI: 10.1126/science.1116480[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvFCisLw%253D&md5=0a7ac2fb7a76c97979d859c0489c682fStructure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with ReceptorLi, Fang; Li, Wenhui; Farzan, Michael; Harrison, Stephen C.Science (Washington, DC, United States) (2005), 309 (5742), 1864-1868CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resoln. of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The at. details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
- 20Gui, M., Song, W., Zhou, H., Xu, J., Chen, S., Xiang, Y., and Wang, X. (2017) Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Res. 27, 119– 129, DOI: 10.1038/cr.2016.152[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFCls7jL&md5=44107ccea29dfcb85c3eba84b999a0c4Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor bindingGui, Miao; Song, Wenfei; Zhou, Haixia; Xu, Jingwei; Chen, Silian; Xiang, Ye; Wang, XinquanCell Research (2017), 27 (1), 119-129CODEN: CREEB6; ISSN:1001-0602. (Nature Publishing Group)The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states detd. by single-particle cryo-electron microscopy. The conformation 1 detd. at 4.3 Å resoln. is three-fold sym. and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 detd. at 7.3, 5.7 and 6.8 Å resolns. are all asym., in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.
- 21Benton, D. J., Wrobel, A. G., Xu, P., Roustan, C., Martin, S. R., Rosenthal, P. B., Skehel, J. J., and Gamblin, S. J. (2020) Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature 588, 327, DOI: 10.1038/s41586-020-2772-0[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1yht7bK&md5=4acff2bd0fa23ff57b0fa46d660772fcReceptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusionBenton, Donald J.; Wrobel, Antoni G.; Xu, Pengqi; Roustan, Chloe; Martin, Stephen R.; Rosenthal, Peter B.; Skehel, John J.; Gamblin, Steven J.Nature (London, United Kingdom) (2020), 588 (7837), 327-330CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain assocd. after cleavage. Fusion activation after receptor binding is proposed to involve the exposure of a 2nd proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide. We analyze the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify 10 different mol. species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer, and dissocd. monomeric S1 bound to ACE2. The 10 structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissocn. of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.
- 22Letko, M., Marzi, A., and Munster, V. (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol 5, 562– 569, DOI: 10.1038/s41564-020-0688-y[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjvFyitL0%253D&md5=98931812734d0f364d4d0c5f88a9d165Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronavirusesLetko, Michael; Marzi, Andrea; Munster, VincentNature Microbiology (2020), 5 (4), 562-569CODEN: NMAICH; ISSN:2058-5276. (Nature Research)Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geog. regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.
- 23Simmons, G., Zmora, P., Gierer, S., Heurich, A., and Pohlmann, S. (2013) Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research. Antiviral Res. 100, 605– 614, DOI: 10.1016/j.antiviral.2013.09.028[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVWksLfK&md5=3585f545c4ce1a19a0ca2f04f2762b44Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral researchSimmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pohlmann, StefanAntiviral Research (2013), 100 (3), 605-614CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)A review. The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.''.
- 24Millet, J. K. and Whittaker, G. R. (2015) Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 202, 120– 134, DOI: 10.1016/j.virusres.2014.11.021[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFyjsbjN&md5=1f9248a8886148418b7f7e6109fd6796Host cell proteases: Critical determinants of coronavirus tropism and pathogenesisMillet, Jean Kaoru; Whittaker, Gary R.Virus Research (2015), 202 (), 120-134CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)A review. Coronaviruses are a large group of enveloped, single-stranded pos.-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a crit. role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential 1st step in establishing infection, the proteolytic activation step is often crit. for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large no. of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the crit. entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range, and pathogenicity.
- 25Madu, I. G., Roth, S. L., Belouzard, S., and Whittaker, G. R. (2009) Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide. J. Virol. 83, 7411– 7421, DOI: 10.1128/JVI.00079-09[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXovFensLk%253D&md5=151e886d42d5f3404d667b00746134c4Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptideMadu, Ikenna G.; Roth, Shoshannah L.; Belouzard, Sandrine; Whittaker, Gary R.Journal of Virology (2009), 83 (15), 7411-7421CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides, while the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a crit. role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochem. analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.
- 26Song, W., Gui, M., Wang, X., and Xiang, Y. (2018) Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog. 14, e1007236, DOI: 10.1371/journal.ppat.1007236[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1Wmtr3M&md5=e0a169e29e03f45d4307941f998df084Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2Song, Wenfei; Gui, Miao; Wang, Xinquan; Xiang, YePLoS Pathogens (2018), 14 (8), e1007236/1-e1007236/19CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding "up" conformation. In addn., we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassocn. of the S1-ACE2 complex from some of the prefusion spikes was obsd. and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state obsd. on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition.
- 27Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., and Veesler, D. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281– 292, DOI: 10.1016/j.cell.2020.02.058[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvVejsLk%253D&md5=ac8a8a208d9c26f88f702fb7634ab1abStructure, Function, and Antigenicity of the SARS-CoV-2 Spike GlycoproteinWalls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, DavidCell (Cambridge, MA, United States) (2020), 181 (2), 281-292.e6CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We detd. cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
- 28Wang, Y., Liu, M., and Gao, J. (2020) Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions. Proc. Natl. Acad. Sci. U. S. A. 117, 13967– 13974, DOI: 10.1073/pnas.2008209117[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVCgsrrN&md5=8c4c5214a202fd3dc7d0ef3008f20be2Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactionsWang, Yingjie; Liu, Meiyi; Gao, JialiProceedings of the National Academy of Sciences of the United States of America (2020), 117 (25), 13967-13974CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Mol. dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein-protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) and the SARS coronavirus in the 2002-2003 (SARS-CoV) outbreak. Anal. of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV-neutralizing antibody 80R are disrupted in the SARS-CoV-2 homol. complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.
- 29Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., and Zhou, Q. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 30Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L. (2020) Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215– 220, DOI: 10.1038/s41586-020-2180-5[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtL8%253D&md5=279c60143e8e5eb505457e0778baa8efStructure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptorLan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, XinquanNature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
- 31Shang, J., Ye, G., Shi, K., Wan, Y., Luo, C., Aihara, H., Geng, Q., Auerbach, A., and Li, F. (2020) Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221– 224, DOI: 10.1038/s41586-020-2179-y[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtbs%253D&md5=33bc9151641b2adcfb0dbf446621a1dcStructural basis of receptor recognition by SARS-CoV-2Shang, Jian; Ye, Gang; Shi, Ke; Wan, Yushun; Luo, Chuming; Aihara, Hideki; Geng, Qibin; Auerbach, Ashley; Li, FangNature (London, United Kingdom) (2020), 581 (7807), 221-224CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystn.) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Addnl., we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
- 32Barh, D., Tiwari, S., Silva Andrade, B., Giovanetti, M., Almeida Costa, E., Kumavath, R., Ghosh, P., Goes-Neto, A., Carlos Junior Alcantara, L., and Azevedo, V. (2020) Potential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain. F1000Research 9, 576, DOI: 10.12688/f1000research.24074.1[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlKqsbzJ&md5=66c465571795a97bc2a9ea7524046f6aPotential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain [version 1; peer review: 2 approved, 1 approved with reservations]Barh, Debmalya; Tiwari, Sandeep; Andrade, Bruno Silva; Giovanetti, Marta; Costa, Eduardo Almeida; Kumavath, Ranjith; Ghosh, Preetam; Goes-Neto, Aristoteles; Alcantara, Luiz Carlos Junior; Azevedo, VascoF1000Research (2020), 9 (), 576CODEN: FRESJL; ISSN:2046-1402. (F1000 Research Ltd.)Background: There are no known medicines or vaccines to control the COVID-19 pandemic caused by SARS-CoV-2 (nCoV). Antiviral peptides are superior to conventional drugs and may also be effective against COVID-19. Hence, we investigated the SARS-CoV-2 Spike receptor-binding domain (nCoV-RBD) that interacts with hACE2 for viral attachment and entry. Methods: Three strategies and bioinformatics approaches were employed to design potential nCoV-RBD - hACE2 interaction-blocking peptides that may restrict viral attachment and entry. Firstly, the key residues interacting with nCoV-RBD - hACE2 are identified and hACE2 sequence-based peptides are designed. Second, peptides from five antibacterial peptide databases that block nCoV-RBD are identified; finally, a chimeric peptide design approach is used to design peptides that can bind to key nCoV-RBD residues. The final peptides are selected based on their physiochem. properties, nos. and positions of key residues binding, binding energy, and antiviral properties. Results: We found that: (i) three amino acid stretches in hACE2 interact with nCoV-RBD; (ii) effective peptides must bind to three key positions of nCoV-RBD (Gly485/Phe486/Asn487, Gln493, and Gln498/Thr500/Asn501); (iii) Phe486, Gln493, and Asn501 are crit. residues; (iv) AC20 and AC23 derived from hACE2 may block two key crit. positions; (iv) DBP6 identified from databases can block the three sites of the nCoV-RBD and interacts with one crit. position, Gln498; (v) seven chimeric peptides were considered promising, among which cnCoVP-3, cnCoVP-4, and cnCoVP-7 are the top three; and (vi) cnCoVP-4 meets all the criteria and is the best peptide. Conclusions: To conclude, using three different bioinformatics approaches, we identified 17 peptides that can potentially bind to the nCoV-RBD that interacts with hACE2. Binding these peptides to nCoV-RBD may potentially inhibit the virus to access hACE2 and thereby may prevent the infection. Out of 17, 10 peptides have promising potential and need further exptl. validation.
- 33Baig, M. S., Alagumuthu, M., Rajpoot, S., and Saqib, U. (2020) Identification of a Potential Peptide Inhibitor of SARS-CoV-2 Targeting its Entry into the Host Cells. Drugs R&D 20, 161– 169, DOI: 10.1007/s40268-020-00312-5
- 34Huang, X., Pearce, R., and Zhang, Y. (2020) De novo design of protein peptides to block association of the SARS-CoV-2 spike protein with human ACE2. Aging 12, 11263– 11276, DOI: 10.18632/aging.103416[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFCmtLnP&md5=45c47fdac442665e6236f468a28f1ed7De novo design of protein peptides to block association of the SARSCoV-2 spike protein with human ACE2Huang, Xiaoqiang; Pearce, Robin; Zhang, YangAging (2020), 12 (12), 11263-11274CODEN: AGINCN; ISSN:1945-4589. (Impact Journals LLC)The outbreak of COVID-19 has now become a global pandemic that has severely impacted lives and economic stability. There is, however, no effective antiviral drug that can be used to treat COVID-19 to date. Built on the fact that SARS-CoV-2 initiates its entry into human cells by the receptor binding domain (RBD) of its spike protein binding to the angiotensin-converting enzyme 2 (hACE2), we extended a recently developed approach, EvoDesign, to design multiple peptide sequences that can competitively bind to the SARS-CoV-2 RBD to inhibit the virus from entering human cells. The protocol starts with the construction of a hybrid peptidic scaffold by linking two fragments grafted from the interface of the hACE2 protein (a.a. 22-44 and 351-357) with a linker glycine, which is followed by the redesign and refinement simulations of the peptide sequence to optimize its binding affinity to the interface of the SARS-CoV-2 RBD. The binding expt. analyses showed that the designed peptides exhibited a significantly stronger binding potency to hACE2 than the wild-type hACE2 receptor (with -53.35 vs. -46.46 EvoEF2 energy unit scores for the top designed and wild-type peptides, resp.). This study demonstrates a new avenue to utilize computationally designed peptide motifs to treat the COVID-19 disease by blocking the crit. spike-RBD and hACE2 interactions.
- 35Han, Y. and Kral, P. (2020) Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2. ACS Nano 14, 5143– 5147, DOI: 10.1021/acsnano.0c02857[ACS Full Text
], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntVKns7s%253D&md5=54088dfed751aff6a9e8dcec95bd25edComputational design of ACE2-based peptide inhibitors of SARS-CoV-2Han, Yanxiao; Kral, PetrACS Nano (2020), 14 (4), 5143-5147CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Peptide inhibitors against the SARS-CoV-2 coronavirus, currently causing a worldwide pandemic, are designed and simulated. The inhibitors are mostly formed by two sequential self-supporting α-helixes (bundle) extd. from the protease domain (PD) of angiotensin-converting enzyme 2 (ACE2), which bind to the SARS-CoV-2 receptor binding domains. Mol. dynamics simulations revealed that the α-helical peptides maintain their secondary structure and provide a highly specific and stable binding (blocking) to SARS-CoV-2. To provide a multivalent binding to the SARS-CoV-2 receptors, many such peptides could be attached to the surfaces of nanoparticle carriers. The proposed peptide inhibitors could provide simple and efficient therapeutics against the COVID-19 disease. - 36Millet, J. K., Tang, T., Nathan, L., Jaimes, J. A., Hsu, H. L., Daniel, S., and Whittaker, G. R. (2019) Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting. J. Visualized Exp. e59010, DOI: 10.3791/59010[Crossref], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlKisLzO&md5=0d8c32a3e9cb66052a32e4ce92bb89f3Production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 settingMillet, Jean K.; Tang, Tiffany; Nathan, Lakshmi; Jaimes, Javier A.; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R.Journal of Visualized Experiments (2019), (145), e59010CODEN: JVEOA4; ISSN:1940-087X. (Journal of Visualized Experiments)The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodol. is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.
- 37Finkelshtein, D., Werman, A., Novick, D., Barak, S., and Rubinstein, M. (2013) LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl. Acad. Sci. U. S. A. 110, 7306– 7311, DOI: 10.1073/pnas.1214441110[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXos1Oitrk%253D&md5=33ff4ae4e6710b0c038db18f715d74d4LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virusFinkelshtein, Danit; Werman, Ariel; Novick, Daniela; Barak, Sara; Rubinstein, MenachemProceedings of the National Academy of Sciences of the United States of America (2013), 110 (18), 7306-7311, S7306/1-S7306/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.
- 38Wang, Y., Grunewald, M., and Perlman, S. (2020) Coronaviruses: An Updated Overview of Their Replication and Pathogenesis. Methods Mol. Biol. 2203, 1– 29, DOI: 10.1007/978-1-0716-0900-2_1[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslSrs73N&md5=2580d7b1adcdc0ab22a87be0352a7442Coronaviruses: an updated overview of their replication and pathogenesisWang, Yuhang; Grunewald, Matthew; Perlman, StanleyMethods in Molecular Biology (New York, NY, United States) (2020), 2203 (Coronaviruses), 1-29CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. Coronaviruses (CoVs), enveloped pos.-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in Dec. 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here the authors provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. The authors will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
- 39Pyrc, K., Berkhout, B., and van der Hoek, L. (2007) Identification of new human coronaviruses. Expert Rev. Anti-Infect. Ther. 5, 245– 253, DOI: 10.1586/14787210.5.2.245[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjs12kt70%253D&md5=a3ae1fbd98bcfa2efab8d5f95b6cd92cIdentification of new human coronavirusesPyrc, Krzysztof; Berkhout, Ben; van der Hoek, LiaExpert Review of Anti-Infective Therapy (2007), 5 (2), 245-253CODEN: ERATCK; ISSN:1478-7210. (Future Drugs Ltd.)A review. To date, there are still a variety of human infections with unknown etiol. Identification of previously unrecognized viral agents in patient samples is of great medical interest but remains a major tech. challenge. Acute respiratory tract infections are responsible for considerable morbidity and mortality in humans and animals. A variety of viruses, bacteria and fungi are assocd. with respiratory tract illness. Most of the respiratory viruses belong to the Paramyxoviridae, Orthomyxoviridae, Picornaviridae, Adenoviridae and Coronaviridae families. No pathogens can be detected in a relatively large proportion of patients with respiratory disease, partially owing to limitations of current diagnostic assays but also since some infections are caused by as yet unknown pathogens. This review will focus on human coronaviruses. In the mid 1960s, two human coronaviruses were identified that cause the common cold: human coronaviruses (HCoV)-229E and HCoV-OC43. The recent outbreak of severe acute respiratory syndrome-CoV and subsequent identification of two addnl. human coronaviruses (HCoV-NL63 and HCoV-HKU1) has drawn attention to this virus family. This review summarizes the knowledge of current methodologies for identifying novel human coronavirus species. Furthermore, information on the discovery of known human coronaviruses will be presented.
- 40Baker, S. C. (2004) Coronaviruses: from common colds to severe acute respiratory syndrome. Pediatr Infect Dis J. 23, 1049– 1050, DOI: 10.1097/01.inf.0000145815.70485.f7[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2crntlaitQ%253D%253D&md5=723b60031a8be06403a1ebb09f51017bCoronaviruses: from common colds to severe acute respiratory syndromeBaker Susan CThe Pediatric infectious disease journal (2004), 23 (11), 1049-50 ISSN:0891-3668.There is no expanded citation for this reference.
- 41Hofmann, H., Pyrc, K., van der Hoek, L., Geier, M., Berkhout, B., and Pohlmann, S. (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc. Natl. Acad. Sci. U. S. A. 102, 7988– 7993, DOI: 10.1073/pnas.0409465102[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvF2isr8%253D&md5=3bd44737b1a64642c5fe7eaaf258b960Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entryHofmann, Heike; Pyrc, Krzysztof; van der Hoek, Lia; Geier, Martina; Berkhout, Ben; Poehlmann, StefanProceedings of the National Academy of Sciences of the United States of America (2005), 102 (22), 7988-7993CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Coronavirus (CoV) infection of humans is usually not assocd. with severe disease. However, discovery of the severe acute respiratory syndrome (SARS) CoV revealed that highly pathogenic human CoVs (HCoVs) can evolve. The identification and characterization of new HCoVs is, therefore, an important task. Recently, a HCoV termed NL63 was discovered in patients with respiratory tract illness. Here, cell tropism and receptor usage of HCoV-NL63 were analyzed. The NL63 spike (S) protein mediated infection of different target cells compared with the closely related 229E-S protein but facilitated entry into cells known to be permissive to SARS-CoV-S-driven infection. An anal. of receptor engagement revealed that NL63-S binds angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV, and HCoV-NL63 uses ACE2 as a receptor for infection of target cells. Potent neutralizing activity directed against NL63- but not 229E-S protein was detected in virtually all sera from patients 8 years of age or older, suggesting that HCoV-NL63 infection of humans is common and usually acquired during childhood. Here, we show that SARS-CoV shares its receptor ACE2 with HCoV-NL63. Because the two viruses differ dramatically in their ability to induce disease, anal. of HCoV-NL63 might unravel pathogenicity factors in SARS-CoV. The frequent HCoV-NL63 infection of humans suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.
- 42Wu, K., Li, W., Peng, G., and Li, F. (2009) Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc. Natl. Acad. Sci. U. S. A. 106, 19970– 19974, DOI: 10.1073/pnas.0908837106[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGjtLrP&md5=f60e9892de280375cc632fc56af4e09eCrystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptorWu, Kailang; Li, Weikai; Peng, Guiqing; Li, FangProceedings of the National Academy of Sciences of the United States of America (2009), 106 (47), 19970-19974, S19970/1-S19970/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). How different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors, was investigated. The crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2 was detd. NL63-CoV RBD has a novel β-sandwich core structure consisting of 2 layers of β-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homol. in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a virus-binding hotspot on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.
- 43Cao, L., Goreshnik, I., Coventry, B., Case, J. B., Miller, L., Kozodoy, L., Chen, R. E., Carter, L., Walls, A. C., Park, Y. J. (2020) De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426– 431, DOI: 10.1126/science.abd9909[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFyqt77I&md5=1d5383cd17badf89367dede22a99cfe9De novo design of picomolar SARS-CoV-2 miniprotein inhibitorsCao, Longxing; Goreshnik, Inna; Coventry, Brian; Case, James Brett; Miller, Lauren; Kozodoy, Lisa; Chen, Rita E.; Carter, Lauren; Walls, Alexandra C.; Park, Young-Jun; Strauch, Eva-Maria; Stewart, Lance; Diamond, Michael S.; Veesler, David; Baker, DavidScience (Washington, DC, United States) (2020), 370 (6515), 426-431CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concn. (IC50) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC50 ∼ 0.16 ng per mL). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.
- 44Larue, R. C., Plumb, M. R., Crowe, B. L., Shkriabai, N., Sharma, A., DiFiore, J., Malani, N., Aiyer, S. S., Roth, M. J., Bushman, F. D. (2014) Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes. Nucleic Acids Res. 42, 4868– 4881, DOI: 10.1093/nar/gku135[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntlykt7g%253D&md5=80a232d3a015225812207c49957def99Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomesLarue, Ross C.; Plumb, Matthew R.; Crowe, Brandon L.; Shkriabai, Nikoloz; Sharma, Amit; Di Fiore, Julia; Malani, Nirav; Aiyer, Sriram S.; Roth, Monica J.; Bushman, Frederic D.; Foster, Mark P.; Kvaratskhelia, MamukaNucleic Acids Research (2014), 42 (8), 4868-4881CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The importance of understanding the mol. mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. The authors have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here the authors show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled the authors to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Addnl., the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant pos. correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. These findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.
- 45McMichael, T. M., Zhang, Y., Kenney, A. D., Zhang, L., Zani, A., Lu, M., Chemudupati, M., Li, J., and Yount, J. S. (2018) IFITM3 Restricts Human Metapneumovirus Infection. J. Infect. Dis. 218, 1582– 1591, DOI: 10.1093/infdis/jiy361[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MboslOksA%253D%253D&md5=12abb01d4838726bc9b5b38fd3450640IFITM3 Restricts Human Metapneumovirus InfectionMcMichael Temet M; Kenney Adam D; Zhang Lizhi; Zani Ashley; Chemudupati Mahesh; Yount Jacob S; McMichael Temet M; Kenney Adam D; Zhang Lizhi; Zani Ashley; Lu Mijia; Chemudupati Mahesh; Li Jianrong; Yount Jacob S; Zhang Yu; Lu Mijia; Li JianrongThe Journal of infectious diseases (2018), 218 (10), 1582-1591 ISSN:.Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.
- 46Hofmann, H., Hattermann, K., Marzi, A., Gramberg, T., Geier, M., Krumbiegel, M., Kuate, S., Uberla, K., Niedrig, M., and Pohlmann, S. (2004) S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J. Virol. 78, 6134– 6142, DOI: 10.1128/JVI.78.12.6134-6142.2004[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkvVequ7s%253D&md5=b0fd1ad9cc7e9b7403acef44dfdd2af8S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patientsHofmann, Heike; Hattermann, Kim; Marzi, Andrea; Gramberg, Thomas; Geier, Martina; Krumbiegel, Mandy; Kuate, Seraphin; Uberla, Klaus; Niedrig, Matthias; Poehlmann, StefanJournal of Virology (2004), 78 (12), 6134-6142CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approx. 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also obsd. with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the anal. of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.
- 47Nahabedian, J., Sharma, A., Kaczmarek, M. E., Wilkerson, G. K., Sawyer, S. L., and Overbaugh, J. (2017) Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entry. Virology 512, 180– 186, DOI: 10.1016/j.virol.2017.09.018[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFyqtrvJ&md5=4606aa191c94d22a307282f94d247258Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entryNahabedian, John; Sharma, Amit; Kaczmarek, Maryska E.; Wilkerson, Greg K.; Sawyer, Sara L.; Overbaugh, JulieVirology (2017), 512 (), 180-186CODEN: VIRLAX; ISSN:0042-6822. (Elsevier B.V.)Studying HIV-1 replication in the presence of functionally related proteins from different species has helped define host determinants of HIV-1 infection. Humans and owl monkeys, but not macaques, encode a CD4 receptor that permits entry of transmissible HIV-1 variants due to a single residue difference. However, little is known about whether divergent CCR5 receptor proteins act as determinants of host-range. Here we show that both owl monkey (Aotus vociferans) CD4 and CCR5 receptors are functional for the entry of transmitted HIV-1 when paired with human versions of the other receptor. By contrast, the owl monkey CD4/CCR5 pair is generally a suboptimal receptor combination, although there is virus-specific variation in infection with owl monkey receptors. Introduction of the human residues 15Y and 16T within a sulfation motif into owl monkey CCR5 resulted in a gain of function. These findings suggest there is cross-talk between CD4 and CCR5 involving the sulfation motif.
- 48Chen, Z., Wang, Y., Ratia, K., Mesecar, A. D., Wilkinson, K. D., and Baker, S. C. (2007) Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63. J. Virol. 81, 6007– 6018, DOI: 10.1128/JVI.02747-06[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmtFGks78%253D&md5=185c639cbd279b519ddd53443afa13c1Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63Chen, Zhongbin; Wang, Yanhua; Ratia, Kiira; Mesecar, Andrew D.; Wilkinson, Keith D.; Baker, Susan C.Journal of Virology (2007), 81 (11), 6007-6018CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is assocd. with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunopptn., and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-assocd. replication complexes. To det. which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.
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Abstract
Figure 1
Figure 1. Dose-dependent inhibition of SARS-CoV-2 Spike-pseudotyped lentivirus infection by SAPs. Dose response curves of the indicated SAPs generated by plotting the percent viral inhibition (y-axis) against the log transformation of SAP concentration (mM, x-axis). Each data point represents the average of three independent experiments, performed in duplicate. Error bars represent standard deviations. The dotted gray line indicates 50% viral inhibition used to determine the IC50 value. Computed IC50 values for the indicated SAPs from three independent experiments ± standard deviations are shown.
Figure 2
Figure 2. Inhibition of Spike- and VSV-G-pseudotyped lentivirus infection by SAPs. Luciferase-encoding lentiviruses pseudotyped with indicated viral glycoprotein were incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of 293T-ACE2 cells. Infection was measured as relative luciferase expression 48 h post-infection. The luciferase signal obtained for the diluent control was set to 100%. Graphs indicate the percentage of infected cells normalized to the diluent control for lentiviruses pseudotyped with (A) SARS-CoV-2 Spike, (B) SARS-CoV Spike, or (C) VSV-G. Bars represent averages from four independent experiments, performed in duplicate, with means from individual experiments shown as circles. Error bars represent standard deviations. Percent infections were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
Figure 3
Figure 3. Binding of SAPs to SARS-CoV-2 Spike. Affinity precipitation of His-tagged SARS-CoV-2 Spike RBD (indicated “His-S RBD”) with FITC-SAPs. (A) Representative SDS-PAGE gels of affinity precipitation of His-S RBD with increasing concentrations of indicated FITC-SAP (lanes 2–8: 0.125, 0.25, 0.5, 1, and 3 mM FITC-SAP). Lane 1 indicates control precipitation of 3 mM FITC-SAP without His-S RBD. FITC-labeled bands were detected at 488 nm fluorescence and His-S RBD was visualized with Coomassie staining. (B) Graphical representation of fluorescence intensities from (A) of indicated FITC-SAP bound to His-S RBD. Each data point represents the average of three independent experiments. Error bars represent standard deviations. Data were fit to the Hill equation to determine the apparent Kd of binding. (C) Calculated binding Kd from three independent experiments ± standard deviations.
Figure 4
Figure 4. Inhibition of SARS-CoV-2 infection by SAPs. SARS-CoV-2 was incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of 293T-ACE2-GFP cells. Infection was measured by flow cytometry as the percentage of cells positive for SARS-CoV-2 nucleocapsid (N) protein 24 h post-infection. (A) Representative flow cytometry plots indicating percent infection. (B) Graph indicates the percentage of infected cells normalized to the diluent control, which was set to 100%. Bars represent averages from two independent experiments, performed in triplicate, with individual data points shown as circles. Error bars represent standard deviations. Percent infections were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
Figure 5
Figure 5. Inhibition of HCoV-NL63 infection by SAPs. HCoV-NL63 was incubated with 3 mM of indicated SAP or diluent control for 1 h prior to infection of LLC-MK2 cells. Cytopathic effects and virus titers in the supernatants were analyzed at 72 h post-infection. (A) Representative bright field microscope images showing cytopathic effects. (B) Graph indicates virus titers in supernatants from LLC-MK2 cells. Bars represent averages from triplicate infections with individual data points shown as circles. Error bars represent standard deviations. Virus titers were compared to the diluent control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. * p < 0.05; ns, not significant.
Figure 6
Figure 6. Graphical illustration of SARS-CoV-2 Spike and SAP6 interaction interface. (A) Overall view of SARS-CoV-2 Spike RBD and human ACE2 interaction mode. The N-terminal helix of human ACE2 is located at the central interface. (B) Relative location of SAP6 (light blue) and SAP1 (green and light blue). (C) H-bond interaction network between SAP6 and SARS-CoV-2 Spike RBD. The Y41, Q42, D38, and E37 of SAP6 peptide are involved in H-bond interactions with T500, Y449, N501, and Y505 of SARS-CoV-2 Spike RBD. Corresponding crystal structure: PDB Code: 6M0J. http://www.rcsb.org/structure/6M0J.
References
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- 1Zhou, P., Yang, X. L., Wang, X. G., Hu, B., Zhang, L., Zhang, W., Si, H. R., Zhu, Y., Li, B., Huang, C. L. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270– 273, DOI: 10.1038/s41586-020-2012-7[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXksFKlsLg%253D&md5=236f17d4d3c7978d72513e5e0258f1b3A pneumonia outbreak associated with a new coronavirus of probable bat originZhou, Peng; Yang, Xing-Lou; Wang, Xian-Guang; Hu, Ben; Zhang, Lei; Zhang, Wei; Si, Hao-Rui; Zhu, Yan; Li, Bei; Huang, Chao-Lin; Chen, Hui-Dong; Chen, Jing; Luo, Yun; Guo, Hua; Jiang, Ren-Di; Liu, Mei-Qin; Chen, Ying; Shen, Xu-Rui; Wang, Xi; Zheng, Xiao-Shuang; Zhao, Kai; Chen, Quan-Jiao; Deng, Fei; Liu, Lin-Lin; Yan, Bing; Zhan, Fa-Xian; Wang, Yan-Yi; Xiao, Geng-Fu; Shi, Zheng-LiNature (London, United Kingdom) (2020), 579 (7798), 270-273CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large no. of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 Dec. 2019, had caused 2,794 lab.-confirmed infections including 80 deaths by 26 Jan. 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence anal. of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addn., 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.
- 2Wu, F., Zhao, S., Yu, B., Chen, Y. M., Wang, W., Song, Z. G., Hu, Y., Tao, Z. W., Tian, J. H., Pei, Y. Y. (2020) A new coronavirus associated with human respiratory disease in China. Nature 579, 265– 269, DOI: 10.1038/s41586-020-2008-3[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXksFKlsLc%253D&md5=0163a684829e880a0c3347e19f0ce52aA new coronavirus associated with human respiratory disease in ChinaWu, Fan; Zhao, Su; Yu, Bin; Chen, Yan-Mei; Wang, Wen; Song, Zhi-Gang; Hu, Yi; Tao, Zhao-Wu; Tian, Jun-Hua; Pei, Yuan-Yuan; Yuan, Ming-Li; Zhang, Yu-Ling; Dai, Fa-Hui; Liu, Yi; Wang, Qi-Min; Zheng, Jiao-Jiao; Xu, Lin; Holmes, Edward C.; Zhang, Yong-ZhenNature (London, United Kingdom) (2020), 579 (7798), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 Jan. 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 Dec. 2019. Epidemiol. investigations have suggested that the outbreak was assocd. with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 Dec. 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic anal. of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.
- 3Sanche, S., Lin, Y. T., Xu, C., Romero-Severson, E., Hengartner, N., and Ke, R. (2020) High Contagiousness and Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging Infect. Dis. 26, 1470– 1477, DOI: 10.3201/eid2607.200282[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlyltr3I&md5=69c7cc5e594d29ac1082e605a2756d0cHigh contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2Sanche, Steven; Lin, Yen Ting; Xu, Chonggang; Romero-Severson, Ethan; Hengartner, Nick; Ke, RuianEmerging Infectious Diseases (2020), 26 (7), 1470-1477CODEN: EIDIFA; ISSN:1080-6059. (Centers for Disease Control and Prevention)Severe acute respiratory syndrome coronavirus 2 is the causative agent of the ongoing coronavirus disease pandemic. Initial ests. of the early dynamics of the outbreak in Wuhan, China, suggested a doubling time of the no. of infected persons of 6-7 days and a basic reproductive no. (R0) of 2.2-2.7. We collected extensive individual case reports across China and estd. key epidemiol. parameters, including the incubation period (4.2 days). We then designed 2 math. modeling approaches to infer the outbreak dynamics in Wuhan by using high-resoln. domestic travel and infection data. Results show that the doubling time early in the epidemic in Wuhan was 2.3-3.3 days. Assuming a serial interval of 6-9 days, we calcd. a median R0 value of 5.7 (95% CI 3.8-8.9). We further show that active surveillance, contact tracing, quarantine, and early strong social distancing efforts are needed to stop transmission of the virus.
- 4Anfinrud, P., Stadnytskyi, V., Bax, C. E., and Bax, A. (2020) Visualizing Speech-Generated Oral Fluid Droplets with Laser Light Scattering. N. Engl. J. Med. 382, 2061– 2063, DOI: 10.1056/NEJMc2007800[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38zls1antg%253D%253D&md5=34b8dfa909b2e5e3fe2082b088dc4131Visualizing Speech-Generated Oral Fluid Droplets with Laser Light ScatteringAnfinrud Philip; Stadnytskyi Valentyn; Bax Adriaan; Bax Christina EThe New England journal of medicine (2020), 382 (21), 2061-2063 ISSN:.There is no expanded citation for this reference.
- 5van Doremalen, N., Bushmaker, T., Morris, D. H., Holbrook, M. G., Gamble, A., Williamson, B. N., Tamin, A., Harcourt, J. L., Thornburg, N. J., Gerber, S. I. (2020) Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. N. Engl. J. Med. 382, 1564– 1567, DOI: 10.1056/NEJMc2004973[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB383ksVKktw%253D%253D&md5=9803ae46c83b19c312f0d810c975378eAerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1van Doremalen Neeltje; Bushmaker Trenton; Holbrook Myndi G; Williamson Brandi N; de Wit Emmie; Munster Vincent J; Morris Dylan H; Gamble Amandine; Tamin Azaibi; Harcourt Jennifer L; Thornburg Natalie J; Gerber Susan I; Lloyd-Smith James OThe New England journal of medicine (2020), 382 (16), 1564-1567 ISSN:.There is no expanded citation for this reference.
- 6Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X. (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497– 506, DOI: 10.1016/S0140-6736(20)30183-5[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Kqu7c%253D&md5=b279b965c1054d99e60f673859c03b49Clinical features of patients infected with 2019 novel coronavirus in Wuhan, ChinaHuang, Chaolin; Wang, Yeming; Li, Xingwang; Ren, Lili; Zhao, Jianping; Hu, Yi; Zhang, Li; Fan, Guohui; Xu, Jiuyang; Gu, Xiaoying; Cheng, Zhenshun; Yu, Ting; Xia, Jiaan; Wei, Yuan; Wu, Wenjuan; Xie, Xuelei; Yin, Wen; Li, Hui; Liu, Min; Xiao, Yan; Gao, Hong; Guo, Li; Xie, Jungang; Wang, Guangfa; Jiang, Rongmeng; Gao, Zhancheng; Jin, Qi; Wang, Jianwei; Cao, BinLancet (2020), 395 (10223), 497-506CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiol., clin., lab., and radiol. characteristics and treatment and clin. outcomes of these patients. All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analyzed data on patients with lab.-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiol. and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. By Jan 2, 2020, 41 admitted hospital patients had been identified as having lab.-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0-58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum prodn. (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0-13·0]). 26 (63%) Of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was assocd. with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiol., duration of human transmission, and clin. spectrum of disease need fulfilment by future studies. Ministry of Science and Technol., Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technol. Commission.
- 7Wang, D., Hu, B., Hu, C., Zhu, F., Liu, X., Zhang, J., Wang, B., Xiang, H., Cheng, Z., Xiong, Y. (2020) Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA 323, 1061– 1069, DOI: 10.1001/jama.2020.1585[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvVSqtbY%253D&md5=2a45d95178b69adc575253dba3e679c2Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, ChinaWang, Dawei; Hu, Bo; Hu, Chang; Zhu, Fangfang; Liu, Xing; Zhang, Jing; Wang, Binbin; Xiang, Hui; Cheng, Zhenshun; Xiong, Yong; Zhao, Yan; Li, Yirong; Wan, Xinghuan; Peng, ZhiyongJAMA, the Journal of the American Medical Association (2020), 323 (11), 1061-1069CODEN: JAMAAP; ISSN:1538-3598. (American Medical Association)In Dec. 2019, novel coronavirus (2019-nCoV)-infected pneumonia (NCIP) occurred in Wuhan, China. The no. of cases has increased rapidly but information on the clin. characteristics of affected patients is limited. A retrospective, single-center case series was performed on the 138 consecutive hospitalized patients with confirmed NCIP at Zhongnan Hospital of Wuhan University in Wuhan, China, from Jan. 1 to Jan. 28, 2020; the final date of follow-up was Feb. 3, 2020. Epidemiol., demog., clin., lab., radiol., and treatment data were collected and analyzed. Outcomes of critically ill patients and noncritically ill patients were compared. Presumed hospital-related transmission was suspected if a cluster of health professionals or hospitalized patients in the same wards became infected and a possible source of infection could be tracked. Of 138 hospitalized patients with NCIP, the median age was 56 yr (interquartile range, 42-68; range, 22-92 yr) and 75 (54.3%) were men. Hospital-assocd. transmission was suspected as the presumed mechanism of infection for affected health professionals (40) and hospitalized patients (17). Common symptoms included fever (136), fatigue (96), and dry cough (82). Lymphopenia (lymphocyte count, 0.8 × 109/L) occurred in 97 patients (70.3%), prolonged prothrombin time (13.0 s) in 80 patients (58%), and elevated lactate dehydrogenase (261 U/L) in 55 patients (39.9%). Chest computed tomog. scans showed bilateral patchy shadows or ground glass opacity in the lungs of all patients. Most patients received antiviral therapy (oseltamivir, 124), and many received antibacterial therapy (moxifloxacin, 89; ceftriaxone, 34; azithromycin, 25) and glucocorticoid therapy (62). Thirty-six patients (26.1%) were transferred to the intensive care unit (ICU) because of complications, including acute respiratory distress syndrome (22), arrhythmia (16), and shock (11). The median time from 1st symptom to dyspnea was 5.0 days, to hospital admission was 7.0 days, and to ARDS was 8.0 days. Patients treated in the ICU (n = 36), compared with patients not treated in the ICU (n = 102), were older (median age, 66 yr vs. 51 yr), were more likely to have underlying comorbidities (26 vs. 38), and were more likely to have dyspnea (23 vs. 20), and anorexia (24 vs. 31). Of the 36 cases in the ICU, 4 (11.1%) received high-flow oxygen therapy, 15 (41.7%) received noninvasive ventilation, and 17 (47.2%) received invasive ventilation (4 were switched to extracorporeal membrane oxygenation). As of Feb. 3, 47 patients (34.1%) were discharged and 6 died (overall mortality, 4.3%), but the remaining patients are still hospitalized. Among those discharged alive (n = 47), the median hospital stay was 10 days (IQR, 7.0-14.0). In this single-center case series of 138 hospitalized patients with confirmed NCIP in Wuhan, China, presumed hospital-related transmission of 2019-nCoV was suspected in 41% of patients, 26% of patients received ICU care, and mortality was 4.3%.
- 8Mao, R., Qiu, Y., He, J. S., Tan, J. Y., Li, X. H., Liang, J., Shen, J., Zhu, L. R., Chen, Y., Iacucci, M. (2020) Manifestations and prognosis of gastrointestinal and liver involvement in patients with COVID-19: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol 5, 667– 678, DOI: 10.1016/S2468-1253(20)30126-6[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38vms12msw%253D%253D&md5=1adb94e19792a53f7a3b2621cc2ace0bManifestations and prognosis of gastrointestinal and liver involvement in patients with COVID-19: a systematic review and meta-analysisMao Ren; Chen Min-Hu; Qiu Yun; He Jin-Shen; Tan Jin-Yu; Li Xue-Hua; Liang Jie; Shen Jun; Zhu Liang-Ru; Chen Yan; Iacucci Marietta; Ghosh Subrata; Ng Siew CThe lancet. Gastroenterology & hepatology (2020), 5 (7), 667-678 ISSN:.BACKGROUND: The prevalence and prognosis of digestive system involvement, including gastrointestinal symptoms and liver injury, in patients with COVID-19 remains largely unknown. We aimed to quantify the effects of COVID-19 on the digestive system. METHODS: In this systematic review and meta-analysis, we systematically searched PubMed, Embase, and Web of Science for studies published between Jan 1, 2020, and April 4, 2020. The websites of WHO, CDC, and major journals were also searched. We included studies that reported the epidemiological and clinical features of COVID-19 and the prevalence of gastrointestinal findings in infected patients, and excluded preprints, duplicate publications, reviews, editorials, single case reports, studies pertaining to other coronavirus-related illnesses, and small case series (<10 cases). Extracted data included author; date; study design; country; patient demographics; number of participants in severe and non-severe disease groups; prevalence of clinical gastrointestinal symptoms such as vomiting, nausea, diarrhoea, loss of appetite, abdominal pain, and belching; and digestive system comorbidities including liver disease and gastrointestinal diseases. Raw data from studies were pooled to determine effect estimates. FINDINGS: We analysed findings from 35 studies, including 6686 patients with COVID-19, that met inclusion criteria. 29 studies (n=6064) reported gastrointestinal symptoms in patients with COVID-19 at diagnosis, and the pooled prevalence of digestive system comorbidities was 4% (95% CI 2-5; range 0-15; I(2)=74%). The pooled prevalence of digestive symptoms was 15% (10-21; range: 2-57; I(2)=96%) with nausea or vomiting, diarrhoea, and loss of appetite being the three most common symptoms. The pooled prevalence of abnormal liver functions (12 studies, n=1267) was 19% (9-32; range 1-53; I(2)=96%). Subgroup analysis showed patients with severe COVID-19 had higher rates of abdominal pain (odds ratio [OR] 7·10 [95% CI 1·93-26·07]; p=0·003; I(2)=0%) and abnormal liver function including increased ALT (1·89 [1·30-2·76]; p=0·0009; I(2)=10%) and increased AST (3·08 [2·14-4·42]; p<0·00001; I(2)=0%) compared with those with non-severe disease. Patients in Hubei province, where the initial COVID-19 outbreak occurred, were more likely to present with abnormal liver functions (p<0·0001) compared with those outside of Hubei. Paediatric patients with COVID-19 had a similar prevalence of gastrointestinal symptoms to those of adult patients. 10% (95% CI 4-19; range 3-23; I(2)=97%) of patients presented with gastrointestinal symptoms alone without respiratory features. Patients who presented with gastrointestinal system involvement had delayed diagnosis (standardised mean difference 2·85 [95% CI 0·22-5·48]; p=0·030; I(2)=73%). Patients with gastrointestinal involvement tended to have a poorer disease course (eg, acute respiratory distress syndrome OR 2·96 [95% CI 1·17-7·48]; p=0·02; I(2)=0%). INTERPRETATION: Our study showed that digestive symptoms and liver injury are not uncommon in patients with COVID-19. Increased attention should be paid to the care of this unique group of patients. FUNDING: None.
- 9Pezzini, A. and Padovani, A. (2020) Lifting the mask on neurological manifestations of COVID-19. Nat. Rev. Neurol. 16, 636– 644, DOI: 10.1038/s41582-020-0398-3[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1OisrbF&md5=7ceecfaa638450e8a5a3e06af1979ff7Lifting the mask on neurological manifestations of COVID-19Pezzini, Alessandro; Padovani, AlessandroNature Reviews Neurology (2020), 16 (11), 636-644CODEN: NRNACP; ISSN:1759-4758. (Nature Research)A review. As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic spreads, it is becoming increasingly evident that coronavirus disease 2019 (COVID-19) is not limited to the respiratory system, and that other organs can be affected. In particular, virus-related neurol. manifestations are being reported more and more frequently in the scientific literature. In this article, we review the literature on the assocn. between COVID-19 and neurol. manifestations, present evidence from preclin. research suggesting that SARS-CoV-2 could be responsible for many of these manifestations, and summarize the biol. pathways that could underlie each neurol. symptom. Understanding the mechanisms that lead to neurol. manifestations in patients with COVID-19 and how these manifestations correlate with clin. outcomes will be instrumental in guiding the optimal use of targeted therapeutic strategies.
- 10Fernandes, A. C. L., Vale, A. J. M., Guzen, F. P., Pinheiro, F. I., Cobucci, R. N., and de Azevedo, E. P. (2020) Therapeutic Options Against the New Coronavirus: Updated Clinical and Laboratory Evidences. Front. Med. 7, 546, DOI: 10.3389/fmed.2020.00546[Crossref], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7gtFentw%253D%253D&md5=491579ab6134dfc2e833957f0c4ea37eTherapeutic Options Against the New Coronavirus: Updated Clinical and Laboratory EvidencesFernandes Amelia Carolina Lopes; Vale Adson Jose Martins; Vale Adson Jose Martins; Guzen Fausto Pierdona; Pinheiro Francisco Irochima; Cobucci Ricardo Ney; de Azevedo Eduardo Pereira; Pinheiro Francisco Irochima; Cobucci Ricardo NeyFrontiers in medicine (2020), 7 (), 546 ISSN:2296-858X.The pandemic caused by the new coronavirus (SARS-Cov-2) has encouraged numerous in vitro studies and clinical trials around the world, with research groups testing existing drugs, novel drug candidates and vaccines that can prevent or treat infection caused by this virus. The urgency for an effective therapy is justified by the easy and fast viral transmission and the high number of patients with severe respiratory distress syndrome who have increasingly occupied intensive care hospital beds, leading to a collapse in health systems in several countries. However, to date, there is no sufficient evidence of the effectiveness of any researched therapy. The off-label or compassionate use of some drugs by health professionals is a reality in all continents, whose permission by regulatory agencies has been based on the results of some clinical trials. In order to guide decision-making for the treatment of COVID-19, this review aims to present studies and guidelines on the main therapies that have been and are currently being tested against SARS-CoV-2 and to critically analyze the reported evidences.
- 11Berlin, D. A., Gulick, R. M., and Martinez, F. J. (2020) Severe Covid-19. N. Engl. J. Med. DOI: 10.1056/NEJMcp2009575
- 12Cui, J., Li, F., and Shi, Z. L. (2019) Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17, 181– 192, DOI: 10.1038/s41579-018-0118-9[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisVyhtbnL&md5=b2b2c40c0cdf30107d8c70f482fc4f63Origin and evolution of pathogenic coronavirusesCui, Jie; Li, Fang; Shi, Zheng-LiNature Reviews Microbiology (2019), 17 (3), 181-192CODEN: NRMACK; ISSN:1740-1526. (Nature Research)A review. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are two highly transmissible and pathogenic viruses that emerged in humans at the beginning of the 21st century. Both viruses likely originated in bats, and genetically diverse coronaviruses that are related to SARS-CoV and MERS-CoV were discovered in bats worldwide. In this Review, we summarize the current knowledge on the origin and evolution of these two pathogenic coronaviruses and discuss their receptor usage; we also highlight the diversity and potential of spillover of bat-borne coronaviruses, as evidenced by the recent spillover of swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs.
- 13Lu, R., Zhao, X., Li, J., Niu, P., Yang, B., Wu, H., Wang, W., Song, H., Huang, B., Zhu, N. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565– 574, DOI: 10.1016/S0140-6736(20)30251-8[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOmsLY%253D&md5=3a644f602ea1c68114e23b5f12625265Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor bindingLu, Roujian; Zhao, Xiang; Li, Juan; Niu, Peihua; Yang, Bo; Wu, Honglong; Wang, Wenling; Song, Hao; Huang, Baoying; Zhu, Na; Bi, Yuhai; Ma, Xuejun; Zhan, Faxian; Wang, Liang; Hu, Tao; Zhou, Hong; Hu, Zhenhong; Zhou, Weimin; Zhao, Li; Chen, Jing; Meng, Yao; Wang, Ji; Lin, Yang; Yuan, Jianying; Xie, Zhihao; Ma, Jinmin; Liu, William J.; Wang, Dayan; Xu, Wenbo; Holmes, Edward C.; Gao, George F.; Wu, Guizhen; Chen, Weijun; Shi, Weifeng; Tan, WenjieLancet (2020), 395 (10224), 565-574CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)In late Dec., 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions detd. by rapid amplification of cDNA ends. Phylogenetic anal. of these 2019-nCoV genomes and those of other coronaviruses was used to det. the evolutionary history of the virus and help infer its likely origin. Homol. modeling was done to explore the likely receptor-binding properties of the virus. The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic anal. revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homol. modeling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues.2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic anal. suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural anal. suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University. These data have been deposited in the ChinaNational Microbiol. Data Center (accession no. NMDC10013002 and genome accession nos. NMDC60013002-01 to NMDC60013002-10) and the datafrom BGI have been deposited in the China National GeneBank (accession nos. CNA000733235).
- 14Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., Graham, B. S., and McLellan, J. S. (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260– 1263, DOI: 10.1126/science.abb2507[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFemt70%253D&md5=27d08cbb9a43d1da051a8a92a9f68aa5Cryo-EM structure of the 2019-nCoV spike in the prefusion conformationWrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
- 15Belouzard, S., Chu, V. C., and Whittaker, G. R. (2009) Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc. Natl. Acad. Sci. U. S. A. 106, 5871– 5876, DOI: 10.1073/pnas.0809524106[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXkvFejtrY%253D&md5=bec80a49cc00c0ebf06a5906ea5c1ccaActivation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sitesBelouzard, Sandrine; Chu, Victor C.; Whittaker, Gary R.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (14), 5871-5876CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a crit. proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.
- 16Simmons, G., Reeves, J. D., Rennekamp, A. J., Amberg, S. M., Piefer, A. J., and Bates, P. (2004) Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc. Natl. Acad. Sci. U. S. A. 101, 4240– 4245, DOI: 10.1073/pnas.0306446101[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFartLw%253D&md5=572fe2967e6bd433308eaf39e27e1930Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entrySimmons, Graham; Reeves, Jacqueline D.; Rennekamp, Andrew J.; Amberg, Sean M.; Piefer, Andrew J.; Bates, PaulProceedings of the National Academy of Sciences of the United States of America (2004), 101 (12), 4240-4245CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addn., we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Addnl., there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.
- 17Shang, J., Wan, Y., Luo, C., Ye, G., Geng, Q., Auerbach, A., and Li, F. (2020) Cell entry mechanisms of SARS-CoV-2. Proc. Natl. Acad. Sci. U. S. A. 117, 11727– 11734, DOI: 10.1073/pnas.2003138117[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1aitL3K&md5=07c0a8dc7eca57b6878841d1441ce8aeCell entry mechanisms of SARS-CoV-2Shang, Jian; Wan, Yushun; Luo, Chuming; Ye, Gang; Geng, Qibin; Auerbach, Ashley; Li, FangProceedings of the National Academy of Sciences of the United States of America (2020), 117 (21), 11727-11734CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochem. and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.
- 18Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280, DOI: 10.1016/j.cell.2020.02.052[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktl2qtb8%253D&md5=60aea5c939a2d4df034a91d6198fb3efSARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease InhibitorHoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, StefanCell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
- 19Li, F., Li, W., Farzan, M., and Harrison, S. C. (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864– 1868, DOI: 10.1126/science.1116480[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvFCisLw%253D&md5=0a7ac2fb7a76c97979d859c0489c682fStructure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with ReceptorLi, Fang; Li, Wenhui; Farzan, Michael; Harrison, Stephen C.Science (Washington, DC, United States) (2005), 309 (5742), 1864-1868CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resoln. of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The at. details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
- 20Gui, M., Song, W., Zhou, H., Xu, J., Chen, S., Xiang, Y., and Wang, X. (2017) Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Res. 27, 119– 129, DOI: 10.1038/cr.2016.152[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFCls7jL&md5=44107ccea29dfcb85c3eba84b999a0c4Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor bindingGui, Miao; Song, Wenfei; Zhou, Haixia; Xu, Jingwei; Chen, Silian; Xiang, Ye; Wang, XinquanCell Research (2017), 27 (1), 119-129CODEN: CREEB6; ISSN:1001-0602. (Nature Publishing Group)The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states detd. by single-particle cryo-electron microscopy. The conformation 1 detd. at 4.3 Å resoln. is three-fold sym. and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 detd. at 7.3, 5.7 and 6.8 Å resolns. are all asym., in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.
- 21Benton, D. J., Wrobel, A. G., Xu, P., Roustan, C., Martin, S. R., Rosenthal, P. B., Skehel, J. J., and Gamblin, S. J. (2020) Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature 588, 327, DOI: 10.1038/s41586-020-2772-0[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1yht7bK&md5=4acff2bd0fa23ff57b0fa46d660772fcReceptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusionBenton, Donald J.; Wrobel, Antoni G.; Xu, Pengqi; Roustan, Chloe; Martin, Stephen R.; Rosenthal, Peter B.; Skehel, John J.; Gamblin, Steven J.Nature (London, United Kingdom) (2020), 588 (7837), 327-330CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain assocd. after cleavage. Fusion activation after receptor binding is proposed to involve the exposure of a 2nd proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide. We analyze the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify 10 different mol. species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer, and dissocd. monomeric S1 bound to ACE2. The 10 structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissocn. of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.
- 22Letko, M., Marzi, A., and Munster, V. (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol 5, 562– 569, DOI: 10.1038/s41564-020-0688-y[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjvFyitL0%253D&md5=98931812734d0f364d4d0c5f88a9d165Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronavirusesLetko, Michael; Marzi, Andrea; Munster, VincentNature Microbiology (2020), 5 (4), 562-569CODEN: NMAICH; ISSN:2058-5276. (Nature Research)Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geog. regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.
- 23Simmons, G., Zmora, P., Gierer, S., Heurich, A., and Pohlmann, S. (2013) Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research. Antiviral Res. 100, 605– 614, DOI: 10.1016/j.antiviral.2013.09.028[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVWksLfK&md5=3585f545c4ce1a19a0ca2f04f2762b44Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral researchSimmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pohlmann, StefanAntiviral Research (2013), 100 (3), 605-614CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)A review. The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.''.
- 24Millet, J. K. and Whittaker, G. R. (2015) Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 202, 120– 134, DOI: 10.1016/j.virusres.2014.11.021[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFyjsbjN&md5=1f9248a8886148418b7f7e6109fd6796Host cell proteases: Critical determinants of coronavirus tropism and pathogenesisMillet, Jean Kaoru; Whittaker, Gary R.Virus Research (2015), 202 (), 120-134CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)A review. Coronaviruses are a large group of enveloped, single-stranded pos.-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a crit. role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential 1st step in establishing infection, the proteolytic activation step is often crit. for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large no. of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the crit. entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range, and pathogenicity.
- 25Madu, I. G., Roth, S. L., Belouzard, S., and Whittaker, G. R. (2009) Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide. J. Virol. 83, 7411– 7421, DOI: 10.1128/JVI.00079-09[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXovFensLk%253D&md5=151e886d42d5f3404d667b00746134c4Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptideMadu, Ikenna G.; Roth, Shoshannah L.; Belouzard, Sandrine; Whittaker, Gary R.Journal of Virology (2009), 83 (15), 7411-7421CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides, while the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a crit. role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochem. analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.
- 26Song, W., Gui, M., Wang, X., and Xiang, Y. (2018) Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog. 14, e1007236, DOI: 10.1371/journal.ppat.1007236[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1Wmtr3M&md5=e0a169e29e03f45d4307941f998df084Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2Song, Wenfei; Gui, Miao; Wang, Xinquan; Xiang, YePLoS Pathogens (2018), 14 (8), e1007236/1-e1007236/19CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding "up" conformation. In addn., we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassocn. of the S1-ACE2 complex from some of the prefusion spikes was obsd. and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state obsd. on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition.
- 27Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., and Veesler, D. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281– 292, DOI: 10.1016/j.cell.2020.02.058[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvVejsLk%253D&md5=ac8a8a208d9c26f88f702fb7634ab1abStructure, Function, and Antigenicity of the SARS-CoV-2 Spike GlycoproteinWalls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, DavidCell (Cambridge, MA, United States) (2020), 181 (2), 281-292.e6CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We detd. cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
- 28Wang, Y., Liu, M., and Gao, J. (2020) Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions. Proc. Natl. Acad. Sci. U. S. A. 117, 13967– 13974, DOI: 10.1073/pnas.2008209117[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVCgsrrN&md5=8c4c5214a202fd3dc7d0ef3008f20be2Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactionsWang, Yingjie; Liu, Meiyi; Gao, JialiProceedings of the National Academy of Sciences of the United States of America (2020), 117 (25), 13967-13974CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Mol. dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein-protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) and the SARS coronavirus in the 2002-2003 (SARS-CoV) outbreak. Anal. of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV-neutralizing antibody 80R are disrupted in the SARS-CoV-2 homol. complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.
- 29Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., and Zhou, Q. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 30Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L. (2020) Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215– 220, DOI: 10.1038/s41586-020-2180-5[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtL8%253D&md5=279c60143e8e5eb505457e0778baa8efStructure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptorLan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, XinquanNature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
- 31Shang, J., Ye, G., Shi, K., Wan, Y., Luo, C., Aihara, H., Geng, Q., Auerbach, A., and Li, F. (2020) Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221– 224, DOI: 10.1038/s41586-020-2179-y[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtbs%253D&md5=33bc9151641b2adcfb0dbf446621a1dcStructural basis of receptor recognition by SARS-CoV-2Shang, Jian; Ye, Gang; Shi, Ke; Wan, Yushun; Luo, Chuming; Aihara, Hideki; Geng, Qibin; Auerbach, Ashley; Li, FangNature (London, United Kingdom) (2020), 581 (7807), 221-224CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystn.) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Addnl., we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
- 32Barh, D., Tiwari, S., Silva Andrade, B., Giovanetti, M., Almeida Costa, E., Kumavath, R., Ghosh, P., Goes-Neto, A., Carlos Junior Alcantara, L., and Azevedo, V. (2020) Potential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain. F1000Research 9, 576, DOI: 10.12688/f1000research.24074.1[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlKqsbzJ&md5=66c465571795a97bc2a9ea7524046f6aPotential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain [version 1; peer review: 2 approved, 1 approved with reservations]Barh, Debmalya; Tiwari, Sandeep; Andrade, Bruno Silva; Giovanetti, Marta; Costa, Eduardo Almeida; Kumavath, Ranjith; Ghosh, Preetam; Goes-Neto, Aristoteles; Alcantara, Luiz Carlos Junior; Azevedo, VascoF1000Research (2020), 9 (), 576CODEN: FRESJL; ISSN:2046-1402. (F1000 Research Ltd.)Background: There are no known medicines or vaccines to control the COVID-19 pandemic caused by SARS-CoV-2 (nCoV). Antiviral peptides are superior to conventional drugs and may also be effective against COVID-19. Hence, we investigated the SARS-CoV-2 Spike receptor-binding domain (nCoV-RBD) that interacts with hACE2 for viral attachment and entry. Methods: Three strategies and bioinformatics approaches were employed to design potential nCoV-RBD - hACE2 interaction-blocking peptides that may restrict viral attachment and entry. Firstly, the key residues interacting with nCoV-RBD - hACE2 are identified and hACE2 sequence-based peptides are designed. Second, peptides from five antibacterial peptide databases that block nCoV-RBD are identified; finally, a chimeric peptide design approach is used to design peptides that can bind to key nCoV-RBD residues. The final peptides are selected based on their physiochem. properties, nos. and positions of key residues binding, binding energy, and antiviral properties. Results: We found that: (i) three amino acid stretches in hACE2 interact with nCoV-RBD; (ii) effective peptides must bind to three key positions of nCoV-RBD (Gly485/Phe486/Asn487, Gln493, and Gln498/Thr500/Asn501); (iii) Phe486, Gln493, and Asn501 are crit. residues; (iv) AC20 and AC23 derived from hACE2 may block two key crit. positions; (iv) DBP6 identified from databases can block the three sites of the nCoV-RBD and interacts with one crit. position, Gln498; (v) seven chimeric peptides were considered promising, among which cnCoVP-3, cnCoVP-4, and cnCoVP-7 are the top three; and (vi) cnCoVP-4 meets all the criteria and is the best peptide. Conclusions: To conclude, using three different bioinformatics approaches, we identified 17 peptides that can potentially bind to the nCoV-RBD that interacts with hACE2. Binding these peptides to nCoV-RBD may potentially inhibit the virus to access hACE2 and thereby may prevent the infection. Out of 17, 10 peptides have promising potential and need further exptl. validation.
- 33Baig, M. S., Alagumuthu, M., Rajpoot, S., and Saqib, U. (2020) Identification of a Potential Peptide Inhibitor of SARS-CoV-2 Targeting its Entry into the Host Cells. Drugs R&D 20, 161– 169, DOI: 10.1007/s40268-020-00312-5
- 34Huang, X., Pearce, R., and Zhang, Y. (2020) De novo design of protein peptides to block association of the SARS-CoV-2 spike protein with human ACE2. Aging 12, 11263– 11276, DOI: 10.18632/aging.103416[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFCmtLnP&md5=45c47fdac442665e6236f468a28f1ed7De novo design of protein peptides to block association of the SARSCoV-2 spike protein with human ACE2Huang, Xiaoqiang; Pearce, Robin; Zhang, YangAging (2020), 12 (12), 11263-11274CODEN: AGINCN; ISSN:1945-4589. (Impact Journals LLC)The outbreak of COVID-19 has now become a global pandemic that has severely impacted lives and economic stability. There is, however, no effective antiviral drug that can be used to treat COVID-19 to date. Built on the fact that SARS-CoV-2 initiates its entry into human cells by the receptor binding domain (RBD) of its spike protein binding to the angiotensin-converting enzyme 2 (hACE2), we extended a recently developed approach, EvoDesign, to design multiple peptide sequences that can competitively bind to the SARS-CoV-2 RBD to inhibit the virus from entering human cells. The protocol starts with the construction of a hybrid peptidic scaffold by linking two fragments grafted from the interface of the hACE2 protein (a.a. 22-44 and 351-357) with a linker glycine, which is followed by the redesign and refinement simulations of the peptide sequence to optimize its binding affinity to the interface of the SARS-CoV-2 RBD. The binding expt. analyses showed that the designed peptides exhibited a significantly stronger binding potency to hACE2 than the wild-type hACE2 receptor (with -53.35 vs. -46.46 EvoEF2 energy unit scores for the top designed and wild-type peptides, resp.). This study demonstrates a new avenue to utilize computationally designed peptide motifs to treat the COVID-19 disease by blocking the crit. spike-RBD and hACE2 interactions.
- 35Han, Y. and Kral, P. (2020) Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2. ACS Nano 14, 5143– 5147, DOI: 10.1021/acsnano.0c02857[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntVKns7s%253D&md5=54088dfed751aff6a9e8dcec95bd25edComputational design of ACE2-based peptide inhibitors of SARS-CoV-2Han, Yanxiao; Kral, PetrACS Nano (2020), 14 (4), 5143-5147CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Peptide inhibitors against the SARS-CoV-2 coronavirus, currently causing a worldwide pandemic, are designed and simulated. The inhibitors are mostly formed by two sequential self-supporting α-helixes (bundle) extd. from the protease domain (PD) of angiotensin-converting enzyme 2 (ACE2), which bind to the SARS-CoV-2 receptor binding domains. Mol. dynamics simulations revealed that the α-helical peptides maintain their secondary structure and provide a highly specific and stable binding (blocking) to SARS-CoV-2. To provide a multivalent binding to the SARS-CoV-2 receptors, many such peptides could be attached to the surfaces of nanoparticle carriers. The proposed peptide inhibitors could provide simple and efficient therapeutics against the COVID-19 disease.
- 36Millet, J. K., Tang, T., Nathan, L., Jaimes, J. A., Hsu, H. L., Daniel, S., and Whittaker, G. R. (2019) Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting. J. Visualized Exp. e59010, DOI: 10.3791/59010[Crossref], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlKisLzO&md5=0d8c32a3e9cb66052a32e4ce92bb89f3Production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 settingMillet, Jean K.; Tang, Tiffany; Nathan, Lakshmi; Jaimes, Javier A.; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R.Journal of Visualized Experiments (2019), (145), e59010CODEN: JVEOA4; ISSN:1940-087X. (Journal of Visualized Experiments)The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodol. is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.
- 37Finkelshtein, D., Werman, A., Novick, D., Barak, S., and Rubinstein, M. (2013) LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl. Acad. Sci. U. S. A. 110, 7306– 7311, DOI: 10.1073/pnas.1214441110[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXos1Oitrk%253D&md5=33ff4ae4e6710b0c038db18f715d74d4LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virusFinkelshtein, Danit; Werman, Ariel; Novick, Daniela; Barak, Sara; Rubinstein, MenachemProceedings of the National Academy of Sciences of the United States of America (2013), 110 (18), 7306-7311, S7306/1-S7306/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.
- 38Wang, Y., Grunewald, M., and Perlman, S. (2020) Coronaviruses: An Updated Overview of Their Replication and Pathogenesis. Methods Mol. Biol. 2203, 1– 29, DOI: 10.1007/978-1-0716-0900-2_1[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslSrs73N&md5=2580d7b1adcdc0ab22a87be0352a7442Coronaviruses: an updated overview of their replication and pathogenesisWang, Yuhang; Grunewald, Matthew; Perlman, StanleyMethods in Molecular Biology (New York, NY, United States) (2020), 2203 (Coronaviruses), 1-29CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. Coronaviruses (CoVs), enveloped pos.-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in Dec. 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here the authors provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. The authors will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
- 39Pyrc, K., Berkhout, B., and van der Hoek, L. (2007) Identification of new human coronaviruses. Expert Rev. Anti-Infect. Ther. 5, 245– 253, DOI: 10.1586/14787210.5.2.245[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjs12kt70%253D&md5=a3ae1fbd98bcfa2efab8d5f95b6cd92cIdentification of new human coronavirusesPyrc, Krzysztof; Berkhout, Ben; van der Hoek, LiaExpert Review of Anti-Infective Therapy (2007), 5 (2), 245-253CODEN: ERATCK; ISSN:1478-7210. (Future Drugs Ltd.)A review. To date, there are still a variety of human infections with unknown etiol. Identification of previously unrecognized viral agents in patient samples is of great medical interest but remains a major tech. challenge. Acute respiratory tract infections are responsible for considerable morbidity and mortality in humans and animals. A variety of viruses, bacteria and fungi are assocd. with respiratory tract illness. Most of the respiratory viruses belong to the Paramyxoviridae, Orthomyxoviridae, Picornaviridae, Adenoviridae and Coronaviridae families. No pathogens can be detected in a relatively large proportion of patients with respiratory disease, partially owing to limitations of current diagnostic assays but also since some infections are caused by as yet unknown pathogens. This review will focus on human coronaviruses. In the mid 1960s, two human coronaviruses were identified that cause the common cold: human coronaviruses (HCoV)-229E and HCoV-OC43. The recent outbreak of severe acute respiratory syndrome-CoV and subsequent identification of two addnl. human coronaviruses (HCoV-NL63 and HCoV-HKU1) has drawn attention to this virus family. This review summarizes the knowledge of current methodologies for identifying novel human coronavirus species. Furthermore, information on the discovery of known human coronaviruses will be presented.
- 40Baker, S. C. (2004) Coronaviruses: from common colds to severe acute respiratory syndrome. Pediatr Infect Dis J. 23, 1049– 1050, DOI: 10.1097/01.inf.0000145815.70485.f7[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2crntlaitQ%253D%253D&md5=723b60031a8be06403a1ebb09f51017bCoronaviruses: from common colds to severe acute respiratory syndromeBaker Susan CThe Pediatric infectious disease journal (2004), 23 (11), 1049-50 ISSN:0891-3668.There is no expanded citation for this reference.
- 41Hofmann, H., Pyrc, K., van der Hoek, L., Geier, M., Berkhout, B., and Pohlmann, S. (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc. Natl. Acad. Sci. U. S. A. 102, 7988– 7993, DOI: 10.1073/pnas.0409465102[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvF2isr8%253D&md5=3bd44737b1a64642c5fe7eaaf258b960Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entryHofmann, Heike; Pyrc, Krzysztof; van der Hoek, Lia; Geier, Martina; Berkhout, Ben; Poehlmann, StefanProceedings of the National Academy of Sciences of the United States of America (2005), 102 (22), 7988-7993CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Coronavirus (CoV) infection of humans is usually not assocd. with severe disease. However, discovery of the severe acute respiratory syndrome (SARS) CoV revealed that highly pathogenic human CoVs (HCoVs) can evolve. The identification and characterization of new HCoVs is, therefore, an important task. Recently, a HCoV termed NL63 was discovered in patients with respiratory tract illness. Here, cell tropism and receptor usage of HCoV-NL63 were analyzed. The NL63 spike (S) protein mediated infection of different target cells compared with the closely related 229E-S protein but facilitated entry into cells known to be permissive to SARS-CoV-S-driven infection. An anal. of receptor engagement revealed that NL63-S binds angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV, and HCoV-NL63 uses ACE2 as a receptor for infection of target cells. Potent neutralizing activity directed against NL63- but not 229E-S protein was detected in virtually all sera from patients 8 years of age or older, suggesting that HCoV-NL63 infection of humans is common and usually acquired during childhood. Here, we show that SARS-CoV shares its receptor ACE2 with HCoV-NL63. Because the two viruses differ dramatically in their ability to induce disease, anal. of HCoV-NL63 might unravel pathogenicity factors in SARS-CoV. The frequent HCoV-NL63 infection of humans suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.
- 42Wu, K., Li, W., Peng, G., and Li, F. (2009) Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc. Natl. Acad. Sci. U. S. A. 106, 19970– 19974, DOI: 10.1073/pnas.0908837106[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGjtLrP&md5=f60e9892de280375cc632fc56af4e09eCrystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptorWu, Kailang; Li, Weikai; Peng, Guiqing; Li, FangProceedings of the National Academy of Sciences of the United States of America (2009), 106 (47), 19970-19974, S19970/1-S19970/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). How different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors, was investigated. The crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2 was detd. NL63-CoV RBD has a novel β-sandwich core structure consisting of 2 layers of β-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homol. in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a virus-binding hotspot on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.
- 43Cao, L., Goreshnik, I., Coventry, B., Case, J. B., Miller, L., Kozodoy, L., Chen, R. E., Carter, L., Walls, A. C., Park, Y. J. (2020) De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426– 431, DOI: 10.1126/science.abd9909[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFyqt77I&md5=1d5383cd17badf89367dede22a99cfe9De novo design of picomolar SARS-CoV-2 miniprotein inhibitorsCao, Longxing; Goreshnik, Inna; Coventry, Brian; Case, James Brett; Miller, Lauren; Kozodoy, Lisa; Chen, Rita E.; Carter, Lauren; Walls, Alexandra C.; Park, Young-Jun; Strauch, Eva-Maria; Stewart, Lance; Diamond, Michael S.; Veesler, David; Baker, DavidScience (Washington, DC, United States) (2020), 370 (6515), 426-431CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concn. (IC50) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC50 ∼ 0.16 ng per mL). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.
- 44Larue, R. C., Plumb, M. R., Crowe, B. L., Shkriabai, N., Sharma, A., DiFiore, J., Malani, N., Aiyer, S. S., Roth, M. J., Bushman, F. D. (2014) Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes. Nucleic Acids Res. 42, 4868– 4881, DOI: 10.1093/nar/gku135[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntlykt7g%253D&md5=80a232d3a015225812207c49957def99Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomesLarue, Ross C.; Plumb, Matthew R.; Crowe, Brandon L.; Shkriabai, Nikoloz; Sharma, Amit; Di Fiore, Julia; Malani, Nirav; Aiyer, Sriram S.; Roth, Monica J.; Bushman, Frederic D.; Foster, Mark P.; Kvaratskhelia, MamukaNucleic Acids Research (2014), 42 (8), 4868-4881CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The importance of understanding the mol. mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. The authors have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here the authors show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled the authors to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Addnl., the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant pos. correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. These findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.
- 45McMichael, T. M., Zhang, Y., Kenney, A. D., Zhang, L., Zani, A., Lu, M., Chemudupati, M., Li, J., and Yount, J. S. (2018) IFITM3 Restricts Human Metapneumovirus Infection. J. Infect. Dis. 218, 1582– 1591, DOI: 10.1093/infdis/jiy361[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MboslOksA%253D%253D&md5=12abb01d4838726bc9b5b38fd3450640IFITM3 Restricts Human Metapneumovirus InfectionMcMichael Temet M; Kenney Adam D; Zhang Lizhi; Zani Ashley; Chemudupati Mahesh; Yount Jacob S; McMichael Temet M; Kenney Adam D; Zhang Lizhi; Zani Ashley; Lu Mijia; Chemudupati Mahesh; Li Jianrong; Yount Jacob S; Zhang Yu; Lu Mijia; Li JianrongThe Journal of infectious diseases (2018), 218 (10), 1582-1591 ISSN:.Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein toll-like receptor 7 (TLR7), and that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.
- 46Hofmann, H., Hattermann, K., Marzi, A., Gramberg, T., Geier, M., Krumbiegel, M., Kuate, S., Uberla, K., Niedrig, M., and Pohlmann, S. (2004) S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J. Virol. 78, 6134– 6142, DOI: 10.1128/JVI.78.12.6134-6142.2004[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkvVequ7s%253D&md5=b0fd1ad9cc7e9b7403acef44dfdd2af8S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patientsHofmann, Heike; Hattermann, Kim; Marzi, Andrea; Gramberg, Thomas; Geier, Martina; Krumbiegel, Mandy; Kuate, Seraphin; Uberla, Klaus; Niedrig, Matthias; Poehlmann, StefanJournal of Virology (2004), 78 (12), 6134-6142CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approx. 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also obsd. with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the anal. of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.
- 47Nahabedian, J., Sharma, A., Kaczmarek, M. E., Wilkerson, G. K., Sawyer, S. L., and Overbaugh, J. (2017) Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entry. Virology 512, 180– 186, DOI: 10.1016/j.virol.2017.09.018[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFyqtrvJ&md5=4606aa191c94d22a307282f94d247258Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entryNahabedian, John; Sharma, Amit; Kaczmarek, Maryska E.; Wilkerson, Greg K.; Sawyer, Sara L.; Overbaugh, JulieVirology (2017), 512 (), 180-186CODEN: VIRLAX; ISSN:0042-6822. (Elsevier B.V.)Studying HIV-1 replication in the presence of functionally related proteins from different species has helped define host determinants of HIV-1 infection. Humans and owl monkeys, but not macaques, encode a CD4 receptor that permits entry of transmissible HIV-1 variants due to a single residue difference. However, little is known about whether divergent CCR5 receptor proteins act as determinants of host-range. Here we show that both owl monkey (Aotus vociferans) CD4 and CCR5 receptors are functional for the entry of transmitted HIV-1 when paired with human versions of the other receptor. By contrast, the owl monkey CD4/CCR5 pair is generally a suboptimal receptor combination, although there is virus-specific variation in infection with owl monkey receptors. Introduction of the human residues 15Y and 16T within a sulfation motif into owl monkey CCR5 resulted in a gain of function. These findings suggest there is cross-talk between CD4 and CCR5 involving the sulfation motif.
- 48Chen, Z., Wang, Y., Ratia, K., Mesecar, A. D., Wilkinson, K. D., and Baker, S. C. (2007) Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63. J. Virol. 81, 6007– 6018, DOI: 10.1128/JVI.02747-06[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmtFGks78%253D&md5=185c639cbd279b519ddd53443afa13c1Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63Chen, Zhongbin; Wang, Yanhua; Ratia, Kiira; Mesecar, Andrew D.; Wilkinson, Keith D.; Baker, Susan C.Journal of Virology (2007), 81 (11), 6007-6018CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is assocd. with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunopptn., and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-assocd. replication complexes. To det. which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.