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Letter
Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond FurinClick to copy article linkArticle link copied!
- Tiffany TangTiffany TangRobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United StatesMore by Tiffany Tang
- Javier A. JaimesJavier A. JaimesDepartment of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, United StatesMore by Javier A. Jaimes
- Miya K. BidonMiya K. BidonRobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United StatesMore by Miya K. Bidon
- Marco R. StrausMarco R. StrausDepartment of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, United StatesMore by Marco R. Straus
- Susan Daniel*Susan Daniel*Email: [email protected]Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United StatesMore by Susan Daniel
- Gary R. Whittaker*Gary R. Whittaker*Email: [email protected]Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, United StatesMore by Gary R. Whittaker
Abstract
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2′ site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.
Copyright © 2021 American Chemical Society
The 21st century has seen the rise of pathogenic strains of human coronaviruses (CoVs) causing major public health concerns, first with the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, (1) then with Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, (2) and now, with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 causes the disease syndrome known as COVID-19, (3) now classified as a pandemic with global reach and impact.
CoV host cell entry is mediated by its spike (S) glycoprotein, a large transmembrane protein that decorates the virus particle. (4) The S protein is demarcated into two domains, the S1 and S2 domains, which contain the receptor binding domain and membrane fusion domain, respectively. There are two proteolytic activation events associated with S-mediated membrane fusion. (5) The first is a priming cleavage that occurs at the interface of the S1 and S2 domain (S1/S2) for some coronaviruses, and the second is the obligatory triggering cleavage that occurs within the S2 region (S2′). (5) The priming cleavage generally converts the S protein into a fusion competent form by enabling the S protein to better bind receptors or expose hidden cleavage sites. (5) The triggering cleavage initiates a series of conformational changes that enable the S protein to harpoon into the host membrane for membrane fusion. (5)
There are a variety of proteases capable of priming and triggering CoV S proteins. While the priming event is not as well characterized and virus dependent, it has been observed that the CoV S can be triggered by proteases at the plasma membrane or endosomal membrane, enabling entry in what are termed “early” and “late” pathways, respectively. (6−8) SARS-CoV was found to utilize the transmembrane bound protease TMPRSS2 to enter using the early pathway. (8−10) However, TMPRSS2 expression is limited to epithelial cell lines, and in TMPRSS2 negative cell lines, SARS-CoV utilizes endosomal cathepsin L to enter using the late pathway. (11) MERS-CoV also utilizes TMPRSS2 and cathepsin L with one major difference. The MERS-CoV S1/S2 boundary contains an RSVR insert that can be recognized by furin or related proprotein convertases (PCs), proteases commonly found in the secretory pathway of most cell lines. (7,12) During the S maturation process, the S protein can be cleaved by furin/PCs. (7,12) Thus, MERS-CoV particles harbor cleaved S1/S2 protein, and it was observed that the S1/S2 priming is crucial for MERS-CoV, (7) but not SARS-CoV, (13) to infect via the early pathway route. However, the S1/S2 cleavage is not a requirement for MERS-CoV late pathway infection. (7)
For SARS-CoV-2, early studies showed that the S1/S2 junction contains an insert with two additional basic residues, P–R–R–A (P, proline; R, arginine; A, alanine), that were not present in SARS-CoV or its closest bat ancestor viruses. (14,15) This insert forms a P–R–R–A–R sequence and while it does contain the minimum furin recognition motif, R–X–X–R, it diverges from the preferred R–X–K/R–R motif because the SARS-CoV-2 sequence has an A in the P3 location instead of an R (16,17) (Figure 1). Intriguingly, the only other known example of this insert on FurinDB, (18) a database of furin substrates, is found in proaerolysin, a bacterial toxin, and was determined to be activated by furin. (19)
Figure 1
Figure 1. Predicted structural model of the SARS-CoV and SARS-CoV-2 S proteins. (Inset) Magnification of the S1/S2 site with conserved R and S residues (red ribbon) and the unique four amino acid insertion P–R–R–A for SARS-CoV-2 (blue ribbon) is shown. The P’s denote the position of that amino acid from the S1/S2 cleavage site, with P1–P5 referring to the amino acids before the cleavage site and P1′ referring to the amino acids after the cleavage site.
Indeed, early studies suggest that SARS-CoV-2 is also processed by furin since SARS-CoV-2 harbors a cleaved S protein, likely due to furin processing at the S1/S2 site. (20−22) Similar to what was observed for MERS-CoV, this S1/S2 cleavage was determined to be a prerequisite for subsequent TMPRSS2 activation at the S2′ site for the infection of respiratory cell lines, such as Calu-3. (23,24) Furthermore, like MERS-CoV, SARS-CoV-2 can also utilize cathepsin L in the endosomal pathway in TMPRSS2-negative cell lines, such as Vero E6. (22)
In this study, we sought to better characterize the SARS-CoV-2 S1/S2 cleavage site and raise some intriguing possibilities for how the site might have emerged. Using pseudoparticles and protease inhibitors, we investigated the impact of S1/S2 cleavage for the successful infection of cells via the early and late pathways. Our data shows that the S1/S2 cleavage is essential for subsequent S2′ activation via TMPRSS2 for entry in Calu-3 cells but not for S2′ activation via cathepsin L for entry in Vero E6 cells and suggests that furin may not be the only protease responsible for the S1/S2 cleavage event.
Results and Discussion
MLV Pseudoparticles as a System to Study SARS-CoV-2 Entry
To assess the functional importance of the S1/S2 site for SARS-CoV-2 entry, we utilized viral pseudoparticles. These particles consist of a murine leukemia virus (MLV) core and are decorated with the viral envelope protein to accurately recapitulate the entry steps of their native counterpart. (25) These particles also contain a luciferase reporter that integrates into the host cell genome upon successful infection and drives the cells to produce luciferase, which is quantifiable. We and others have used MLV-based pseudoparticles widely to study CoV entry pathways. (12,21,26) Using the HEK293T cell line, MLV pseudoparticles containing the wild-type SARS-CoV-2 S protein (SARS-CoV-2pp) or the SARS-CoV S protein (SARS-CoVpp) were generated alongside positive control particles containing the vesicular stomatitis virus G protein (VSVpp) or negative control particles lacking envelope proteins (Δenvpp).
Since coronaviruses can enter via the “early” or “late” pathway, we chose to infect cell lines representative of each pathway, as the entry mechanism can be highly cell-type dependent. (27) We utilized the Calu-3 (early) and the Vero E6 (late) cell lines for these studies, which activate the SARS-CoV-2 S2′ using TMPRSS2 and cathepsin L, respectively. As expected, VSVpp (positive control) infected Vero E6 and Calu-3 cells with several orders of magnitude higher luciferase units than the values reported with Δenvpp infection (negative control) (Figure 2A,B). This confirms that the envelope protein is driving infection and not the particle itself. In the case of SARS-CoVpp and SARS-CoV-2pp, both particles are infectious in both cell lines as they drive luciferase production several orders of magnitude higher than Δenvpp (Figure 2A,B) with SARS-CoVpp more infectious than SARS-CoV-2pp.
Figure 2
Figure 2. Characterization of an MLVpp system. (A, B) MLVpp infectivity in Vero E6 and Calu-3 cells. Cells were infected with MLVpps exhibiting the SARS-CoV-2 S, SARS-CoV S, VSV G, or no envelope protein and assessed for luciferase activity. Error bars represent the standard error measurements of three biological replicates (n = 3). (C) Western blot analysis of SARS-CoV-2pp and SARS-CoVpp S content. S was detected using a rabbit antibody against the SARS-CoV-2 S2 region and cross reacts against the SARS-CoV S. MLV content was detected using a mouse antibody against MLV p30. The image was cropped from a singular Western blot.
The pseudoparticles were also probed for their S content via Western blot. For the SARS-CoV-2pp, we detected a band at 85 kDa and for SARS-CoVpp, we detected a strong band at 185 kDa (Figure 2C). The different bands observed between SARS-CoV-2pp and SARS-CoVpp are likely because SARS-CoV-2 S is cleaved at the S1/S2 location by furin/PCs during biosynthesis, whereas SARS-CoV S cannot be cleaved by furin/PCs. Thus, the 85 kDa band corresponds to the S2 segment (S2) of the SARS-CoV-2 S as the primary antibody recognizes the S2 region, while the 185 kDa band corresponds to the uncleaved (S0) SARS-CoV S. We note that both SARS-CoVpp and SARS-CoV-2pp exhibit bands at >185 kDa that likely correspond to dimeric and trimeric forms of S. These oligomeric forms of S have been reported in other SARS-CoV-2pp Western blots. (22,28) Since SARS-CoVpp harbors full-length S, its oligomeric S bands are of higher molecular weight than the SARS-CoV-2pp oligomeric S bands, which harbor cleaved S. Furthermore, the higher order bands may be more apparent due to the use of a polyclonal antibody to detect both the SARS-CoV and SARS-CoV-2 S.
Use of the dec-RVKR-CMK Protease Inhibitor to Produce SARS-CoV-2pp with Uncleaved S
To examine the functional role of S priming by furin/PC proteases, we needed to produce SARS-CoV-2pp harboring uncleaved S protein. This can be accomplished by treating producer cells with an appropriate protease inhibitor to prevent S1/S2 cleavage during biosynthesis. We chose dec-RVKR-CMK because it has been shown to inhibit furin/PCs, preventing S1/S2 cleavage. (23,26,29) Furthermore, we have previously showed that the addition of dec-RVKR-CMK to producer cells generates MLV pseudotyped particles that harbor full-length MERS-CoV S protein (MERS-CoVpp), (26) and we observe a similar trend with SARS-CoV-2pp generated from cells treated with dec-RVKR-CMK (Figure 3C, lanes 1 and 2), exhibiting full-length uncleaved S0 and weak cleaved S2 bands. As mentioned, the additional bands at >185 kDa likely correspond to dimeric and trimeric S, as previously reported in other Western blots probing for SARS-CoV-2pp S, (22,28) with the higher order bands from particles produced in cells treated with dec-RVKR-CMK observed at higher molecular weights.
Figure 3
Figure 3. Impact of dec-RVKR-CMK on MLVpps. + dec-RVKR-CMK refers to MLVpp produced in HEK293T treated with 75 μM dec-RVKR-CMK at the time of transfection. + furin refers to particles treated with 6 U of recombinant furin for 3 h at 37 °C. (A, B) ± dec-RVKR-CMK and ± furin SARS-CoV-2pp were used to infect Vero E6 and Calu-3 cells. Infectivity was normalized to the – dec-RVKR-CMK, – furin condition in each cell line. Note the change in y-axis scale between A and B. Error bars represent the standard error measurements of three biological replicates (n = 3). Statistical analysis was performed using an unpaired student’s t test. Not shown: there is a ns difference between the −dec-RVKR-CMK, ± furin conditions (black bars). *, P ≤ 0.05; **, P ≤ 0.01; ns, nonsignificant, P > 0.05. (C) Western blot analysis of SARS-CoV-2pp produced in ± dec-RVKR-CMK and treated with ± furin for S protein content to complement the infection conditions. S was detected using a rabbit antibody against the SARS-CoV-2 S2 region. MLV content was detected using a mouse antibody against MLV p30. Band intensities were normalized to the MLV p30 band intensity.
The dec-RVKR-CMK-treated (uncleaved) particles were used to transduce cells to observe the impact of S1/S2 cleavage for subsequent S2′ activation via early acting TMPRSS2 in Calu-3 cells or late acting cathepsin L in Vero E6 cells. In Vero E6 cells, the uncleaved SARS-CoV-2pp were 30-fold more infectious than their cleaved counterparts, suggesting that uncleaved particles result in more infectious particles and that the S1/S2 cleavage is not required or hinders infection via the late pathway (Figure 3A). However, in Calu-3 cells, the opposite trend was observed; the uncleaved particles were significantly less infectious than their cleaved counterparts (Figure 3B), suggesting that S1/S2 cleavage is essential (23,24) for early entry. As a control, we also generated SARS-CoVpp, which lacks the furin/PC S1/S2 site, in the presence or absence of dec-RVKR-CMK and noted no significant impact of dec-RVKR-CMK on SARS-CoVpp infectivity in both Vero E6 and Calu-3 cells (Figure S1A,B). This affirms that the dec-RVKR-CMK impacts on infectivity are due to blocking the S1/S2 cleavage.
Thus, we were interested to see if we could restore the infection of the uncleaved particles by treating them with exogeneous furin, which has been previously shown to process purified SARS-CoV-2 S protein. (30) In Calu-3, we observed a 2-fold increase in infection when uncleaved particles were treated with exogeneous furin, but with cleaved particles, we did not observe any statistically significant increase with furin treatment (Figure 3B). In Vero E6 cells, for both types of particles, we did not observe a statistically significant increase in infection with furin treatment (Figure 3A).
Since furin treatment only modestly increased infectivity in all cases observed, we wanted to visualize how efficiently furin was processing the SARS-CoV-2 S via Western blot. For cleaved particles, exogeneous furin processed the faint full-length S0 band into S2 (Figure 3C, lanes 1 and 3). This trend was confirmed upon quantifying the intensities of the S0 and S2 bands relative to the intensity of the MLV p30 band; furin treatment reduced the relative S0 intensity from 0.35 to 0.02, resulting in an increase in relative S2 intensity from 1.74 to 2.83. For uncleaved particles, furin treatment had a more modest impact, as the relative intensity of the S0 bands was reduced from 2.61 to 2.25, resulting in a small increase in relative S2 intensity from 0.66 to 0.92 (Figure 3C, lanes 2 and 4).
The Western blot bands also reveal that the relative intensity of the uncleaved SARS-CoV-2pp S0 bands is greater than the relative intensity of the cleaved SARS-CoV-2pp S2 bands, and this trend was previously observed with MERS-CoVpp S (26) (Figure 3C, lanes 1 and 2), but not with SARS-CoV (Figure S1C). This likely suggests that the increased infectivity we observed with the uncleaved SARS-CoV-2pp in Vero E6 cells could be due to particles harboring a greater amount of S protein. A possible explanation of why uncleaved particles could harbor more S is because uncleaved S is more stable than cleaved S, as S1/S2 cleavage reduces S stability. (30) Thus, cleavage may encourage greater S degradation, reducing the amount of S available for incorporation into the pseudoparticles. However, we did not observe an increased infectivity with uncleaved SARS-CoV-2pp in Calu-3 cells because of the S1/S2 cleavage requirement, and an increased S content cannot compensate for cleavage.
Due to the limited ability of exogenous furin to rescue and cleave SARS-CoV-2 S, we re-evaluated the role of furin in processing the S1/S2 site. Since dec-RVKR-CMK can also inhibit a number of furin related PCs, the effects that have been observed with dec-RVKR-CMK on particles could have resulted from the inhibition of other PC proteases and not necessarily furin. Therefore, we produced particles in the presence of alpha1-PDX, a more selective furin inhibitor (31) than dec-RVKR-CMK. Infectivity results of these particles in Vero E6 cells show that the alpha1-PDX furin specific inhibitor failed to recapitulate the high level of enhancement provided by dec-RVKR-CMK at all tested inhibitor concentrations (Figure 4A). This indicates that dec-RVKR-CMK may have also been acting on non-furin proteases that can also process the S1/S2 site and furin itself may not be the only protease processing the S1/S2 site, as is generally assumed.
Figure 4
Figure 4. Use of specific furin (alpha1-PDX) and broad furin (dec-RVKR-CMK) inhibitors to investigate furin specificity. (A) Infectivity of particles produced in the presence of broad (dec-RVKR-CMK) and specific (alpha1-PDX) furin inhibitors. The inhibitor was applied at the indicated concentration to producer HEK293T cells. Particles were used to infect Vero E6 cells. Infectivity was normalized to the – condition. Error bars represent the standard error measurements of four biological replicates (n = 4). Statistical analysis was performed using an unpaired student’s t test compared to the – condition. *, P ≤ 0.05; ns, nonsignificant, P > 0.05. (B) Proteolytic cleavage assay of SARS-CoV-2 S1/S2 peptides to confirm alpha1-PDX and dec-RVKR-CMK inhibition specificity. Alpha1-PDX (2 μM), dec-RVKR-CMK (75 μM), or no inhibitor was incubated with either furin or trypsin recombinant protease and a fluorogenic peptide mimicking the SARS-CoV-2 S1/S2 site (TNSPRRARSVA). Results represent averages, and error bars represent the standard error measurement of three biological replicates (n = 3).
As a control, the activity of the alpha1-PDX was confirmed using a proteolytic cleavage assay, in which proteolytic cleavage of fluorogenic peptides results in a fluorescence increase used to determine the velocity of the cleavage reaction (Vmax). We have previously used this assay to observe furin cleavage of fluorogenic peptides mimicking the SARS-CoV-2 S1/S2 site (TNSPRRARSVA). (32) The addition of the alpha1-PDX or dec-RVKR-CMK inhibitor to a peptide reaction including furin resulted in almost complete inhibition of the activity, as very limited cleavage was detected. Interestingly, the addition of the alpha1-PDX or dec-RVKR-CMK to a reaction including trypsin gave different results. While alpha1-PDX did modestly inhibit the trypsin-mediated cleavage, dec-RVKR-CMK exhibited almost complete inhibition of trypsin activity. Thus, the results of this peptide cleavage assay confirm that alpha1-PDX is active on furin-like substrates and is a much more specific inhibitor compared to dec-RVKR-CMK (Figure 4B).
Our published results demonstrated that the PC protease PC1 can process SARS-CoV-2 S1/S2, (32) which opens up the possibility that non-furin proteases within the secretory pathway in producer cells can process the SARS-CoV-2 “furin-like” S1/S2 site, as originally suggested by Coutard et al. (33) In the studies reported here, we utilize HEK293T cells to produce the pseudoparticles, which may express a different repertoire of proteases than those in clinically relevant cells. In the future, it will be important to investigate the impact of S1/S2 processing by non-furin PCs in cells infected with SARS-CoV-2.
Furin-Cleavage Predictions of the SARS-CoV-2 S1/S2 Site
To gain further insights on furin processing of the S1/S2 sites, we utilized the PiTou (34) and ProP (35) cleavage prediction tools to analyze the likelihood that the S1/S2 site is processed by furin. ProP predicts furin cleavage sites based on networks derived from experimental data, whereas PiTou uses a combination of a hidden Markov model and biological knowledge-based cumulative probability score functions to characterize a 20 amino acid motif from P14 to P6′ that reflects the binding strength and accessibility of the motif in the furin binding pocket. Thus, the PiTou algorithm has been reported to be more sensitive and specific than ProP for predicting furin cleavage. (34) Both algorithms agree with each other in predicting furin cleavage, with positive scores (PiTou) and scores above 0.5 (ProP) indicating furin cleavage, strengthening the predictions (Figure 5), but since the PiTou algorithm is more sensitive and specific, we decided to focus on the PiTou values for further analysis.
Figure 5
Figure 5. Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV S sequences were analyzed using the ProP (35) 1.0 and PiTou (34) 3.0 furin prediction algorithm. Bold scores indicate the S1/S2 sequence is predicted to be cleaved by furin. Red box highlights SARS-CoV and SARS-CoV-2 scores for reader’s convenience. Purple lines denote the position of the predicted S1/S2 cleavage site. Basic arginine (R) and lysine (K) residues are highlighted in blue. *For Bat-RmYN02, the sequence number was determined from the S alignment with SARS-CoV-2 S using Geneious. Sequences corresponding to the S1/S2 region of HCoV-HKU1 (AAT98580.1), SARS-CoV (AAT74874.1), SARS-CoV-2 (QHD43416.1), Bat-CoVRaTG13 (QHR63300.2), Bat-SL-CoVZC45 (AVP78031.1), Bat-SL-CoVZXC21 (AVP78042.1), MERS-CoV (AFS88936.1), BatCoV-HKU4 (YP_001039953.1), BatCoV-HKU5 (YP_001039962.1), BatCoV-PML (KC869678), Bat-CoV-HKU9 (YP_001039971), and Influenza A/Chicken/Hong Kong/822.1/01/H5N1 (AF509026.2) were obtained from GenBank. The sequence corresponding to the S1/S2 region of RmYN02 (EPI_ISL_412977) was obtained from GISAID.
The PiTou algorithm predicts that the SARS-CoV-2 S1/S2 site (PiTou score: 9.2) can be cleaved by furin, whereas other lineage B-betaCoVs that have been proposed as SARS-CoV-2 precursors, such as SARS-CoV, RaTG13, ZC45, ZXC21, and RmYN02, are predicted not to be cleaved by furin. Notably, the PiTou scores of traditionally accepted furin cleavage sites, such as those found in influenza H5N1 (PiTou score: 13.6) and HCoV-HKU1 (PiTou score: 14.6), are much higher than the score for SARS-CoV-2, suggesting that while the SARS-CoV-2 S1/S2 site has increased furin recognition when compared to other lineage B-betaCoVs, the site is not optimal for furin cleavage, in alignment with the experimental data shown here suggesting poor furin specific cleavage.
The modest SARS-CoV-2 PiTou score raises the question about the origin and evolution of the S1/S2 site of the virus. It is generally believed that the SARS-CoV-2 S1/S2 site is an insertion of “polybasic” residues, (36) as occurs for avian influenza virus, but as the identity of the SARS-CoV-2 ancestor virus still remains to be discovered, it is important to explore alternative explanations. It has recently been proposed that unsampled SARS-like CoV lineages(s) may be circulating in bats, which are important for the evolutionary origins of SARS-CoV-2. (37) Of note, a “breakpoint sequence hypothesis” has been proposed for the introduction of a furin-like cleavage site at S1/S2, (38) an idea further elucidated by Lytras et al. (39) in the context of the origin of SARS-like viruses. We consider that SARS-CoV-2 could have originated via recombination with a currently unknown ancestor bat virus with a robust furin cleavage site (i.e., PiTou score: approximately 13–15), but one which cannot bind to a human receptor, and that in order to gain the ability to infect human cells, the furin cleavage site may have been downregulated.
These suggested parallels are informed by lineage C-betaCoV in which MERS-CoV has a modest furin score (PiTou score: 5.2), but its closest known ancestor bat viruses, BatCoV-HKU4 (PiTou score: < 0), BatCoV-HKU5 (PiTou score: 10.3), and BatCoV-PML (PiTou score: 2.175), exhibit a wide range of furin scores. Of note is that the virus with the highest furin score, BatCoV-HKU5 (PiTou score: 10.3), cannot bind the MERS-CoV hDPP4 receptor, whereas BatCoV-HKU4 (PiTou score: <0) can, (40) so the MERS-CoV S1/S2 site can be seen as a downregulation from BatCoV-HKU5 to bind hDPP4.
Overall, our results support observations (23,24) that the role of this “gained” SARS-CoV-2 S1/S2 site is to expand viral tropism to respiratory cells, likely in the upper airways, allowing efficient transmission. The cleaved S1/S2 site is crucial for the SARS-CoV-2 S to be subsequently cleaved at the S2′ location by TMPRSS2 for early entry in respiratory cells. Without the S1/S2 cleavage, we predict that SARS-CoV-2 would be endocytosed and, due to low cathepsin L expression in respiratory endosomes, (7) SARS-CoV-2 would not efficiently infect via this route. If SARS-CoV-2 infects TMPRSS2 negative cells, it can utilize endosomal cathepsin L, an ubiquitous protease generally found throughout mammalian cells, (41) to activate the S protein, (22) though at undetermined sites. However, for cathepsin L activation, S1/S2 cleavage is not required, and our results indicate that preventing this cleavage increases the infectivity of SARS-CoV-2, at least in Vero E6 cells. This may be connected to recent work showing that S1/S2 cleavage reduces the thermal stability of SARS-CoV-2 S, (30) and by preventing this cleavage, there is more S available for incorporation into the pseudoparticles. Interestingly, the S1/S2 site activation appears to also have a role in the immune response against SARS-CoV-2, as it has been shown that SARS-CoV-2 S with a deleted S1/S2 loop can provide a better protective immune response than that with the S1/S2 loop. (42)
The role of furin in activating the S1/S2 site was also investigated. As protease inhibitors commonly employed in cell entry studies, such as dec-RVKR-CMK, can also inhibit other proteases in addition to furin, (29) it is difficult to ascertain the impact of individual proteases. While furin likely processes the SARS-CoV-2 site, our data suggests that other PCs are also involved, since Western blots show poor processing of uncleaved S upon the addition of purified furin and the use of a highly selective furin inhibitor (alpha1-PDX) has a modest impact on infectivity. All in all, as we consider protease-based inhibitors in treating COVID-19, especially those targeting furin, (43,44) it is important to thoroughly evaluate the role of these proteases to be certain that the relevant proteases are targeted.
Methods
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Predicted Structural Modeling
Cells, Plasmids, and Reagents
HEK293T (accession no: CRL-11268, ATCC), Vero E6 (accession no: CRL-1586, ATCC), and Calu-3 (accession no: HTB-55) cells were maintained in an incubator at 37 °C and 5% CO2 and cultivated in Dulbecco’s Modified Eagle Medium (Cellgro) supplemented with 10% Hyclone FetalClone II (GE) and 10 mM HEPES (Cellgro) (DMEMc). Cells were passaged using Dulbecco’s phosphate-buffered saline (DPBS) and Trypsin EDTA (1×; Cellgro). The pCMV-MLV-gagpol murine leukemia virus (MLV) packaging construct, the pTG-luc luciferase reporter, pCAGGS/VSV-G, pCAGGS, and pcDNA/SARS-S plasmids are as previously described. (25) The pcDNA/SARS2-S was a generous gift from the Veesler lab. (21)
Protease inhibitor decanoyl-RVKR-CMK (dec-RVKR-CMK) was purchased from Tocris and resuspended in sterile water to 10 mM. Recombinant furin was purchased from New England Biolabs. Recombinant l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin was obtained from Sigma-Aldrich. Human alpha-1 PDX (alpha1-PDX) recombinant protein was purchased from ThermoFisher Scientific.
The SARS-CoV-2 S1/S2 fluorogenic peptide with the sequence TNSPRRARSVA, sandwiched between the (7-methoxycoumarin-4-yl)acetyl/2,4-dinitrophenyl (MCA/DNP) FRET pair, was synthesized by Biomatik.
Furin Prediction Calculations
ProP
CoV sequences were input into the ProP 1.0 Server hosted here: http://www.cbs.dtu.dk/services/ProP/.
PiTou
CoV sequences were analyzed using the PiTou V3 software freely available via request. (34)
Pseudoparticle Production
Pseudotyped viruses were produced from published protocols with minor modifications. (25) Briefly, HEK293T cells were seeded to 30% confluency. Cells were transfected with pTG-luc (600 ng), pCMV-MLV-gagpol (800 ng), and the respective viral envelope protein (600 ng) using polyethylenimine for 48 h. Supernatants were then harvested, centrifuged, clarified, and stored in −80 °C aliquots.
For + dec-RVKR-CMK particles, 7.5 μL of dec-RVKR-CMK was added to cells immediately after transfection and boosted with an additional 7.5 μL 24 h later (final concentration: 75 μM).
For alpha1-PDX particles, the indicated concentrations were added to cells immediately after transfection, as recommended by the manufacturer.
Pseudoparticle Assays
Infection assays were as previously described with minor modifications (25) in triplicate. Briefly, target cells were seeded to confluency. Cells were washed with DPBS, infected with particles, and incubated on a rocker for 1.5 h. Cells were supplemented with DMEMc and incubated for 72 h. Cells were lysed, and luciferase activity was assessed using the Luciferase Assay System and the Glomax 20/20 luminometer (Promega). Infectivity values were analyzed and plotted using Prism 8.
Exogeneous Protease Treatment
One milliliter of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 h at 42 000 rpm and 4 °C. Particles were resuspended in 30 μL of DPBS buffer supplemented with 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Particles were treated with 6 U of recombinant furin for 3 h at 37 °C.
Western Blot Analysis of Pseudoparticles
Three milliliters of pseudoparticles was pelleted and resuspended as described above, but in 20 μL. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to the samples, which were then heated at 65 °C for 20 min. Samples were separated on a NuPAGE Bis-Tris gel (Invitrogen) and transferred to polyvinylidene difluoride membranes (GE). SARS-CoV-2 and SARS-CoV S were detected using a rabbit polyclonal antibody that recognizes the S2 domain (Cat: 40590-T62, Sinobiological) and an AlexaFluor 488 goat anti-rabbit secondary antibody. MLV protein was detected using a mouse monoclonal antibody that recognizes the MLV p30 protein (Cat: ab130757, Abcam) and an AlexaFluor 488 goat anti-mouse secondary antibody. Bands were detected using the ChemiDoc Imaging software (Bio-Rad). S0 and S2 band intensity was calculated using the analysis tools on Biorad Image Lab 6.1 software and normalized to the MLV p30 band intensity.
Fluorogenic Peptide Assay
Peptide assays were as previously described with minor modifications (32) in triplicates. Each reaction was performed in a 100 μL volume consisting of buffer, protease, inhibitor, and SARS-CoV-2 S1/S2 fluorogenic peptide in an opaque 96-well plate. For furin catalyzed reactions, 1 U/well recombinant furin was diluted in buffer consisting of 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol. For trypsin catalyzed reactions, 0.8 nM/well TPCK trypsin was diluted in PBS buffer. The alpha1-PDX inhibitor or dec-RVKR-CMK inhibitor was added at a concentration of 2 or 75 μM, respectively. Peptide was added last at a concentration of 50 μM.
Fluorescence emission was measured every minute for 60 min using a SpectraMax fluorometer (Molecular Devices) at 30 °C with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Vmax was calculated by fitting the linear rise in fluorescence to the equation of a line.
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Impact of the dec-RVKR-CMK inhibitor on SARS-CoVpp production, which lacks the furin S1/S2 site (PDF)
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Author Information
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- Susan Daniel - Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States;
http://orcid.org/0000-0001-7773-0835;
- Tiffany Tang - Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States;http://orcid.org/0000-0002-5130-5248
- Javier A. Jaimes - Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, United States;http://orcid.org/0000-0001-6706-092X
Acknowledgments
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This work was funded by the National Institute of Health research grant R01AI35270 and Fast Grant, Mercatus Center. T.T. acknowledges support by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441 and the Samuel C. Fleming Family Graduate Fellowship. We would especially like to thank Jean Millet for providing important insight on analyzing PiTou values and calculations for BatCoV-HKU9. We would like to thank Hector Aguilar-Carreno for helpful input and thank members of the Daniel and Whittaker groups as well as the Eliezer and Weinstein groups at Weill Cornell for helpful discussions. S.D. acknowledges funding for this project, sponsored by the Defense Advanced Research Projects Agency (DARPA) Army Research Office and accomplished under Cooperative Agreement Number W911NF-18-2-0152. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of DARPA or the Army Research Office or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein.
References
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This article references 44 other publications.
- 1Zhong, N. S., Zheng, B. J., Li, Y. M., Poon, L. L. M., Xie, Z. H., Chan, K. H., Li, P. H., Tan, S. Y., Chang, Q., Xie, J. P., Liu, X. Q., Xu, J., Li, D. X., Yuen, K. Y., Peiris, J. S. M., and Guan, Y. (2003) Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People’s Republic of China, in February, 2003. Lancet 362, 1353– 1358, DOI: 10.1016/S0140-6736(03)14630-2Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3srhtVKgug%253D%253D&md5=eacca67e582fec65edccfb6b5cec0e14Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003Zhong N S; Zheng B J; Li Y M; Poon; Xie Z H; Chan K H; Li P H; Tan S Y; Chang Q; Xie J P; Liu X Q; Xu J; Li D X; Yuen K Y; Peiris; Guan YLancet (London, England) (2003), 362 (9393), 1353-8 ISSN:.BACKGROUND: An epidemic of severe acute respiratory syndrome (SARS) has been associated with an outbreak of atypical pneumonia originating in Guangdong Province, People's Republic of China. We aimed to identify the causative agent in the Guangdong outbreak and describe the emergence and spread of the disease within the province. METHODS: We analysed epidemiological information and collected serum and nasopharyngeal aspirates from patients with SARS in Guangdong in mid-February, 2003. We did virus isolation, serological tests, and molecular assays to identify the causative agent. FINDINGS: SARS had been circulating in other cities of Guangdong Province for about 2 months before causing a major outbreak in Guangzhou, the province's capital. A novel coronavirus, SARS coronavirus (CoV), was isolated from specimens from three patients with SARS. Viral antigens were also directly detected in nasopharyngeal aspirates from these patients. 48 of 55 (87%) patients had antibodies to SARS CoV in their convalescent sera. Genetic analysis showed that the SARS CoV isolates from Guangzhou shared the same origin with those in other countries, and had a phylogenetic pathway that matched the spread of SARS to the other parts of the world. INTERPRETATION: SARS CoV is the infectious agent responsible for the epidemic outbreak of SARS in Guangdong. The virus isolated from patients in Guangdong is the prototype of the SARS CoV in other regions and countries.
- 2Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D. M. E., and Fouchier, R. A. M. (2012) Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. N. Engl. J. Med. 367, 1814– 1820, DOI: 10.1056/NEJMoa1211721Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1ekt73P&md5=4fc960f8008c5a4b76a08fbdeb224ac8Isolation of a novel coronavirus from a man with pneumonia in Saudi ArabiaZaki, Ali Moh; van Boheemen, Sander; Bestebroer, Theo M.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.New England Journal of Medicine (2012), 367 (19), 1814-1820CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)A previously unknown coronavirus was isolated from the sputum of a 60-yr-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clin. data, virus isolation, and mol. identification are presented. The clin. picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
- 3Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., Gao, G. F., and Tan, W. (2020) A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 382, 727– 733, DOI: 10.1056/NEJMoa2001017Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjslGmsrc%253D&md5=73cc5c839e1e934da69b39537063b7b3A novel coronavirus from patients with pneumonia in China, 2019Zhu, Na; Zhang, Dingyu; Wang, Wenling; Li, Xingwang; Yang, Bo; Song, Jingdong; Zhao, Xiang; Huang, Baoying; Shi, Weifeng; Lu, Roujian; Niu, Peihua; Zhan, Faxian; Ma, Xuejun; Wang, Dayan; Xu, Wenbo; Wu, Guizhen; Gao, George F.; Tan, WenjieNew England Journal of Medicine (2020), 382 (8), 727-733CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)In Dec. 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. Complete genome sequences of the three novel coronaviruses were submitted to GISAID (BetaCoV/Wuhan/ IVDC-HB-01/2019, accession ID: EPI_ISL_402119; BetaCoV/Wuhan/IVDC-HB-04/2020, accession ID: EPI_ISL_402120; BetaCoV/Wuhan/IVDC-HB-05/2019, accession ID: EPI_ISL_402121).
- 4Belouzard, S., Millet, J. K., Licitra, B. N., and Whittaker, G. R. (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4, 1011– 1033, DOI: 10.3390/v4061011Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpvFyltrk%253D&md5=327223944a652c715b0a1550d0e4038aMechanisms of coronavirus cell entry mediated by the viral spike proteinBelouzard, Sandrine; Millet, Jean K.; Licitra, Beth N.; Whittaker, Gary R.Viruses (2012), 4 (), 1011-1033CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)A review. Coronaviruses are enveloped pos.-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes - A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
- 5White, J. M. and Whittaker, G. R. (2016) Fusion of Enveloped Viruses in Endosomes. Traffic 17, 593– 614, DOI: 10.1111/tra.12389Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlslWls70%253D&md5=915c967ab0f18e75e824f6cfc3de5cdfFusion of enveloped viruses in endosomesWhite, Judith M.; Whittaker, Gary R.Traffic (Oxford, United Kingdom) (2016), 17 (6), 593-614CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)A review. Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biol. in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. The authors end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid compn.) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion.
- 6Heald-Sargent, T. and Gallagher, T. (2012) Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence. Viruses 4, 557– 580, DOI: 10.3390/v4040557Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslOqsbk%253D&md5=68753d01793151eea573e5e90a853877Ready, set, fuse! the coronavirus spike protein and acquisition of fusion competenceHeald-Sargent, Taylor; Gallagher, TomViruses (2012), 4 (), 557-580CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)A review. Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies.
- 7Park, J. E., Li, K., Barlan, A., Fehr, A. R., Perlman, S., McCray, P. B., and Gallagher, T. (2016) Proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism. Proc. Natl. Acad. Sci. U. S. A. 113, 12262– 12267, DOI: 10.1073/pnas.1608147113Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1elur3F&md5=25764c2eb9d46c8ab09f70c461f8cb95Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropismPark, Jung-Eun; Li, Kun; Barlan, Arlene; Fehr, Anthony R.; Perlman, Stanley; McCray, Paul B. Jr.; Gallagher, TomProceedings of the National Academy of Sciences of the United States of America (2016), 113 (43), 12262-12267CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus-cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely det. how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.
- 8Shulla, A., Heald-Sargent, T., Subramanya, G., Zhao, J., Perlman, S., and Gallagher, T. (2011) A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry Downloaded from. J. Virol. 85, 873– 882, DOI: 10.1128/JVI.02062-10Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXisVOktLg%253D&md5=3dd5d16397b36bda4d781044694321bdA transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entryShulla, Ana; Heald-Sargent, Taylor; Subramanya, Gitanjali; Zhao, Jinchun; Perlman, Stanley; Gallagher, TomJournal of Virology (2011), 85 (2), 873-882CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunopptn. captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a sep. endoprotease operates as a portal for activation of SARS-CoV cell entry.
- 9Matsuyama, S., Nagata, N., Shirato, K., Kawase, M., Takeda, M., and Taguchi, F. (2010) Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. J. Virol. 84, 12658– 12664, DOI: 10.1128/JVI.01542-10Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlsVejtA%253D%253D&md5=01c4cb62576a1047d696d1712c68bdbaEfficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2Matsuyama, Shutoku; Nagata, Noriyo; Shirato, Kazuya; Kawase, Miyuki; Takeda, Makoto; Taguchi, FumihiroJournal of Virology (2010), 84 (24), 12658-12664CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, prodn. of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.
- 10Glowacka, I., Bertram, S., Müller, M. A., Allen, P., Soilleux, E., Pfefferle, S., Steffen, I., Tsegaye, T. S., He, Y., Gnirss, K., Niemeyer, D., Schneider, H., Drosten, C., and Pöhlmann, S. (2011) Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response. J. Virol. 85, 4122– 4134, DOI: 10.1128/JVI.02232-10Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MvjvF2ntA%253D%253D&md5=94e4268b223e658c2b592d2e1cf19839Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune responseGlowacka Ilona; Bertram Stephanie; Muller Marcel A; Allen Paul; Soilleux Elizabeth; Pfefferle Susanne; Steffen Imke; Tsegaye Theodros Solomon; He Yuxian; Gnirss Kerstin; Niemeyer Daniela; Schneider Heike; Drosten Christian; Pohlmann StefanJournal of virology (2011), 85 (9), 4122-34 ISSN:.The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.
- 11Simmons, G., Gosalia, D. N., Rennekamp, A. J., Reeves, J. D., Diamond, S. L., and Bates, P. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc. Natl. Acad. Sci. U. S. A. 102, 11876– 11881, DOI: 10.1073/pnas.0505577102Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsFGgurw%253D&md5=99f26bc819463a5377187542db9bdfe5Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entrySimmons, Graham; Gosalia, Dhaval N.; Rennekamp, Andrew J.; Reeves, Jacqueline D.; Diamond, Scott L.; Bates, PaulProceedings of the National Academy of Sciences of the United States of America (2005), 102 (33), 11876-11881CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currently no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) glycoprotein, and infection is sensitive to lysosomotropic agents that perturb endosomal pH. We demonstrate here that the lysosomotropic-agent-mediated block to SARS-CoV infection is overcome by protease treatment of target-cell-assocd. virus. In addn., SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. A cell-free membrane-fusion system demonstrates that engagement of receptor followed by proteolysis is required for SARS-CoV membrane fusion and indicates that cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These results suggest that SARS-CoV infection results from a unique, three-step process: receptor binding and induced conformational changes in S glycoprotein followed by cathepsin L proteolysis within endosomes. The requirement for cathepsin L proteolysis identifies a previously uncharacterized class of inhibitor for SARS-CoV infection.
- 12Millet, J. K. and Whittaker, G. R. (2014) Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. Proc. Natl. Acad. Sci. U. S. A. 111, 15214– 15219, DOI: 10.1073/pnas.1407087111Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1yhtb7E&md5=21bea9924d802dd56de1dac9d38bd509Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike proteinMillet, Jean Kaoru; Whittaker, Gary R.Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (42), 15214-15219CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2' position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2' site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus.
- 13Reinke, L. M., Spiegel, M., Plegge, T., Hartleib, A., Nehlmeier, I., Gierer, S., Hoffmann, M., Hofmann-Winkler, H., Winkler, M., and Pöhlmann, S. (2017) Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS One 12 (6), e0179177 DOI: 10.1371/journal.pone.0179177Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFynt7Y%253D&md5=b4e700c4fed413eb6f5b4358b1cf010dDifferent residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Poehlmann, StefanPLoS One (2017), 12 (6), e0179177/1-e0179177/15CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.
- 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.abb2507Google 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.
- 15Jaimes, J. A., André, N. M., Chappie, J. S., Millet, J. K., and Whittaker, G. R. (2020) Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J. Mol. Biol. 432, 3309– 3325, DOI: 10.1016/j.jmb.2020.04.009Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnvFeju78%253D&md5=887ae39cca30bb371f36d7980c4c76d1Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation LoopJaimes, Javier A.; Andre, Nicole M.; Chappie, Joshua S.; Millet, Jean K.; Whittaker, Gary R.Journal of Molecular Biology (2020), 432 (10), 3309-3325CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) originally arose as part of a major outbreak of respiratory disease centered on Hubei province, China. It is now a global pandemic and is a major public health concern. Taxonomically, SARS-CoV-2 was shown to be a Betacoronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2). Subsequently, betacoronaviruses from pangolins were identified as close relatives to SARS-CoV-2. Here, we perform structural modeling of the SARS-CoV-2 spike glycoprotein. Our data provide support for the similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, we identify an extended structural loop contg. basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains. We suggest this loop confers fusion activation and entry properties more in line with betacoronaviruses in lineages A and C, and be a key component in the evolution of SARS-CoV-2 with this structural loop affecting virus stability and transmission.
- 16Henrich, S., Lindberg, I., Bode, W., and Than, M. E. (2005) Proprotein convertase models based on the crystal structures of furin and kexin: Explanation of their specificity. J. Mol. Biol. 345, 211– 227, DOI: 10.1016/j.jmb.2004.10.050Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2cnjs1Sgtw%253D%253D&md5=667d12e3a21b22016d05d9108ff2bd33Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificityHenrich Stefan; Lindberg Iris; Bode Wolfram; Than Manuel EJournal of molecular biology (2005), 345 (2), 211-27 ISSN:0022-2836.In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.
- 17Henrich, S., Cameron, A., Bourenkov, G. P., Kiefersauer, R., Huber, R., Lindberg, I., Bode, W., and Than, M. E. (2003) The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat. Struct. Mol. Biol. 10, 520– 526, DOI: 10.1038/nsb941Google ScholarThere is no corresponding record for this reference.
- 18Tian, S., Huang, Q., Fang, Y., and Wu, J. (2011) FurinDB: A Database of 20-residue furin cleavage site motifs, substrates and their associated drugs. Int. J. Mol. Sci. 12, 1060– 1065, DOI: 10.3390/ijms12021060Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXitlSntb8%253D&md5=6baad48472fc42c9b938183c34102fffFurinDB: a database of 20-residue furin cleavage site motifs, substrates and their associated drugsTian, Sun; Huang, Qingsheng; Fang, Ying; Wu, JianhuaInternational Journal of Molecular Sciences (2011), 12 (), 1060-1065CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)FurinDB (freely available online) is a database of furin substrates. This database includes exptl. verified furin cleavage sites, substrates, species, exptl. methods, original publications of expts. and assocd. drugs targeting furin substrates. The current database release contains 126 furin cleavage sites from three species: mammals, bacteria and viruses. A main feature of this database is that all furin cleavage sites are recorded as a 20-residue motif, including one core region (eight amino acids, P6-P2') and two flanking solvent accessible regions (eight amino acids, P7-P14, and four amino acids, P3'-P6'), that represent our current understanding of the mol. biol. of furin cleavage. This database is important for understanding the mol. evolution and relationships between sequence motifs, 3D structures, cellular functions and phys. properties required by furin for cleavage, and for elucidating the mol. mechanisms and the progression of furin cleavage assocd. human diseases, including pathogenic infections, neurol. disorders, tumorigenesis, tumor invasion, angiogenesis, and metastasis. FurinDB database will be a solid addn. to the publicly available infrastructure for scientists in the field of mol. biol.
- 19Abrami, L., Fivaz, M., Decroly, E., Seidah, N. G., Jean, F., Thomas, G., Leppla, S. H., Buckley, J. T., and Van Der Goot, F. G. (1998) The pore-forming toxin proaerolysin is activated by furin. J. Biol. Chem. 273, 32656– 32661, DOI: 10.1074/jbc.273.49.32656Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnvFOntb4%253D&md5=3aa5f2f2855acb2901c6890841478e68The pore-forming toxin proaerolysin is activated by furinAbrami, Laurence; Fivaz, Marc; Decroly, Etienne; Seidah, Nabil G.; Jean, Francois; Thomas, Gary; Leppla, Stephen H.; Buckley, J. Thomas; Van Der Goot, F. GisouJournal of Biological Chemistry (1998), 273 (49), 32656-32661CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor α1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.
- 20Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A., Müller, M. A., Drosten, C., and Pöhlmann, S. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280.E8, DOI: 10.1016/j.cell.2020.02.052Google Scholar20https://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.
- 21Walls, 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, DOI: 10.1016/j.cell.2020.02.058Google Scholar21https://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.
- 22Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J., and Qian, Z. (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620, DOI: 10.1038/s41467-020-15562-9Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFyjt78%253D&md5=6b0b1ef5a68f4a35da4aabecb0f99544Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoVOu, Xiuyuan; Liu, Yan; Lei, Xiaobo; Li, Pei; Mi, Dan; Ren, Lili; Guo, Li; Guo, Ruixuan; Chen, Ting; Hu, Jiaxin; Xiang, Zichun; Mu, Zhixia; Chen, Xing; Chen, Jieyong; Hu, Keping; Jin, Qi; Wang, Jianwei; Qian, ZhaohuiNature Communications (2020), 11 (1), 1620CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
- 23Hoffmann, M., Kleine-Weber, H., and Pöhlmann, S. (2020) A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol. Cell 78, 779– 784.E5, DOI: 10.1016/j.molcel.2020.04.022Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovVCgs7o%253D&md5=18c626bd22d3d9c280d8b5e19c116db8A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung CellsHoffmann, Markus; Kleine-Weber, Hannah; Poehlmann, StefanMolecular Cell (2020), 78 (4), 779-784.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site contg. multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
- 24Bestle, D., Heindl, M. R., Limburg, H., Van Lam van, T., Pilgram, O., Moulton, H., Stein, D. A., Hardes, K., Eickmann, M., Dolnik, O., Rohde, C., Klenk, H. D., Garten, W., Steinmetzer, T., and Böttcher-Friebertshäuser, E. (2020) TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci. alliance 3, e202000786, DOI: 10.26508/lsa.202000786Google ScholarThere is no corresponding record for this reference.
- 25Millet, 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. 2019, e59010, DOI: 10.3791/59010Google ScholarThere is no corresponding record for this reference.
- 26Straus, M. R., Tang, T., Lai, A. L., Flegel, A., Bidon, M., Freed, J. H., Daniel, S., and Whittaker, G. R. (2020) Ca 2+ ions promote fusion of Middle East respiratory syndrome coronavirus with host cells and increase infectivity. J. Virol. 94, 426– 446, DOI: 10.1128/JVI.00426-20Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKhurrF&md5=d8475cfbe72278344793d2ff2aff99abCa2+ ions promote fusion of middle east respiratory syndrome coronavirus with host cells and increase infectivityStraus, Marco R.; Tang, Tiffany; Lai, Alex L.; Flegel, Annkatrin; Bidon, Miya; Freed, Jack H.; Daniel, Susan; Whittaker, Gary R.Journal of Virology (2020), 94 (13), e00426-20CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Fusion with, and subsequent entry into, the host cell is one of the crit. steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homol., and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approx. 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent ESR (ESR) expts. revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approx. 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.
- 27Tang, T., Bidon, M., Jaimes, J. A., Whittaker, G. R., and Daniel, S. (2020) Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res. 178, 104792, DOI: 10.1016/j.antiviral.2020.104792Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsFalt7w%253D&md5=08a629b31ad760c3bf19c2175abe2363Coronavirus membrane fusion mechanism offers a potential target for antiviral developmentTang, Tiffany; Bidon, Miya; Jaimes, Javier A.; Whittaker, Gary R.; Daniel, SusanAntiviral Research (2020), 178 (), 104792CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)A review. The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapies against the causative agent, SARS-CoV-2, and also against other pathogenic coronaviruses (CoV) that have emerged in the past or might appear in future. Researchers are therefore focusing on steps in the CoV replication cycle that may be vulnerable to inhibition by broad-spectrum or specific antiviral agents. The conserved nature of the fusion domain and mechanism across the CoV family make it a valuable target to elucidate and develop pan-CoV therapeutics. In this article, we review the role of the CoV spike protein in mediating fusion of the viral and host cell membranes, summarizing the results of research on SARS-CoV, MERS-CoV, and recent peer-reviewed studies of SARS-CoV-2, and suggest that the fusion mechanism be investigated as a potential antiviral target. We also provide a supplemental file contg. background information on the biol., epidemiol., and clin. features of all human-infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.
- 28Hyseni, I., Molesti, E., Benincasa, L., Piu, P., Casa, E., Temperton, N. J., Manenti, A., and Montomoli, E. (2020) Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays. Viruses 12, 1011, DOI: 10.3390/v12091011Google ScholarThere is no corresponding record for this reference.
- 29Matsuyama, S., Shirato, K., Kawase, M., Terada, Y., Kawachi, K., Fukushi, S., and Kamitani, W. (2018) Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells. J. Virol. 92, e00683-18, DOI: 10.1128/JVI.00683-18Google ScholarThere is no corresponding record for this reference.
- 30Wrobel, A. G., Benton, D. J., Xu, P., Roustan, C., Martin, S. R., Rosenthal, P. B., Skehel, J. J., and Gamblin, S. J. (2020) SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 27, 763– 767, DOI: 10.1038/s41594-020-0468-7Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlGlurfM&md5=75e5b3c971677ffd82d98090b57255ecSARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effectsWrobel, Antoni G.; Benton, Donald J.; Xu, Pengqi; Roustan, Chloe; Martin, Stephen R.; Rosenthal, Peter B.; Skehel, John J.; Gamblin, Steven J.Nature Structural & Molecular Biology (2020), 27 (8), 763-767CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. We investigate the relation of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We detd. cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochem. characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and ∼1000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
- 31Jean, F., Stella, K., Thomas, L., Liu, G., Xiang, Y., Reason, A. J., and Thomas, G. (1998) α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent. Proc. Natl. Acad. Sci. U. S. A. 95, 7293– 7298, DOI: 10.1073/pnas.95.13.7293Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXktFChtr0%253D&md5=af8752ea7d2cac8404c0b858a9f7d6a4α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agentJean, Francois; Stella, Kori; Thomas, Laurel; Liu, Gseping; Xiang, Yang; Reason, Andrew J.; Thomas, GaryProceedings of the National Academy of Sciences of the United States of America (1998), 95 (13), 7293-7298CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The important role of furin in the proteolytic activation of many pathogenic mols. has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochem. anal. of the specificity of a bacterially expressed His-and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin mols. and functions as a suicide substrate inhibitor. Once bound to furin's active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center-Arg355-Ile-Pro-Arg358- or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small mol. furin inhibitors.
- 32Jaimes, J. A., Millet, J. K., and Whittaker, G. R. (2020) Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site. iScience 23, 101212, DOI: 10.1016/j.isci.2020.101212Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFahur7I&md5=fb4f2beee3529e458165e526f75377f8Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 SiteJaimes, Javier A.; Millet, Jean K.; Whittaker, Gary R.iScience (2020), 23 (6), 101212CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochem. evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.
- 33Coutard, B., Valle, C., de Lamballerie, X., Canard, B., Seidah, N. G., and Decroly, E. (2020) The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res. 176, 104742, DOI: 10.1016/j.antiviral.2020.104742Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjtFertr0%253D&md5=144e90f8ac4dc1a517c899b10c4a7a98The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same cladeCoutard, B.; Valle, C.; de Lamballerie, X.; Canard, B.; Seidah, N. G.; Decroly, E.Antiviral Research (2020), 176 (), 104742CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.
- 34Tian, S., Huajun, W., and Wu, J. (2012) Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseases. Sci. Rep. 2, 261, DOI: 10.1038/srep00261Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC383ltl2ktQ%253D%253D&md5=e9467e011fabed34fe6da3a913697d35Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseasesTian Sun; Huajun Wang; Wu JianhuaScientific reports (2012), 2 (), 261 ISSN:.Furin cleaves diverse types of protein precursors in the secretory pathway. The substrates for furin cleavage possess a specific 20-residue recognition sequence motif. In this report, based on the functional characterisation of the 20-residue sequence motif, we developed a furin cleavage site prediction tool, PiTou, using a hybrid method composed of a hidden Markov model and biological knowledge-based cumulative probability score functions. PiTou can accurately predict the presence and location of furin cleavage sites in protein sequences with high sensitivity (96.9%) and high specificity (97.3%). PiTou's prediction scores are biological meaningful and reflect binding strength and solvent accessibility of furin substrates. A prediction result is interpreted within cellular contexts: subcellular localisation, cellular function and interference by other dynamic protein modifications. Combining next-generation sequencing, PiTou can help with elucidating the molecular mechanism of furin cleavage-associated human diseases. PiTou has been made freely available at the associated website.
- 35Duckert, P., Brunak, S., and Blom, N. (2004) Prediction of proprotein convertase cleavage sites. Protein Eng., Des. Sel. 17, 107– 112, DOI: 10.1093/protein/gzh013Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlKgsbw%253D&md5=042a34ce1e8a987079be99bf0f60f104Prediction of proprotein convertase cleavage sitesDuckert, Peter; Brunak, Soren; Blom, NikolajProtein Engineering, Design & Selection (2004), 17 (1), 107-112CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)Many secretory proteins and peptides are synthesized as inactive precursors that, in addn. to signal peptide cleavage, undergo post-translational processing to become biol. active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created addnl. focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on exptl. results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network.
- 36Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C., and Garry, R. F. (2020) The proximal origin of SARS-CoV-2. Nat. Med. 26, 450– 452, DOI: 10.1038/s41591-020-0820-9Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXltFCjtbY%253D&md5=3489259c33e29365c0c1cf7fc5613407The proximal origin of SARS-CoV-2Andersen, Kristian G.; Rambaut, Andrew; Lipkin, W. Ian; Holmes, Edward C.; Garry, Robert F.Nature Medicine (New York, NY, United States) (2020), 26 (4), 450-452CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)There is no expanded citation for this reference.
- 37Boni, M. F., Lemey, P., Jiang, X., Lam, T. T. Y., Perry, B. W., Castoe, T. A., Rambaut, A., and Robertson, D. L. (2020) Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. Nat. Microbiol. 5, 1408– 1417, DOI: 10.1038/s41564-020-0771-4Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVyjt7vP&md5=d9469eec71c82371598422c09bb5534bEvolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemicBoni, Maciej F.; Lemey, Philippe; Jiang, Xiaowei; Lam, Tommy Tsan-Yuk; Perry, Blair W.; Castoe, Todd A.; Rambaut, Andrew; Robertson, David L.Nature Microbiology (2020), 5 (11), 1408-1417CODEN: NMAICH; ISSN:2058-5276. (Nature Research)Abstr.: There are outstanding evolutionary questions on the recent emergence of human coronavirus SARS-CoV-2 including the role of reservoir species, the role of recombination and its time of divergence from animal viruses. We find that the sarbecoviruses-the viral subgenus contg. SARS-CoV and SARS-CoV-2-undergo frequent recombination and exhibit spatially structured genetic diversity on a regional scale in China. SARS-CoV-2 itself is not a recombinant of any sarbecoviruses detected to date, and its receptor-binding motif, important for specificity to human ACE2 receptors, appears to be an ancestral trait shared with bat viruses and not one acquired recently via recombination. To employ phylogenetic dating methods, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Bayesian evolutionary rate and divergence date ests. were shown to be consistent for these three approaches and for two different prior specifications of evolutionary rates based on HCoV-OC43 and MERS-CoV. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estd. as 1948 (95% highest posterior d. (HPD): 1879-1999), 1969 (95% HPD: 1930-2000) and 1982 (95% HPD: 1948-2009), indicating that the lineage giving rise to SARS-CoV-2 has been circulating unnoticed in bats for decades.
- 38Gallaher, W. R. (2020) A palindromic RNA sequence as a common breakpoint contributor to copy-choice recombination in SARS-COV-2. Arch. Virol. 165, 2341– 2348, DOI: 10.1007/s00705-020-04750-zGoogle Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFShu7bN&md5=29efb730adb00258d37baeea1ebf0a50A palindromic RNA sequence as a common breakpoint contributor to copy-choice recombination in SARS-COV-2Gallaher, William R.Archives of Virology (2020), 165 (10), 2341-2348CODEN: ARVIDF; ISSN:0304-8608. (Springer-Verlag GmbH)Much remains unknown concerning the origin of the novel pandemic coronavirus that has raged across the globe since emerging in Wuhan of Hubei province, near the center of the People's Republic of China, in Dec. of 2019. All current members of the family Coronaviridae have arisen by a combination of incremental adaptive mutations, against the backdrop of many recombinational events throughout the past, rendering each a unique mosaic of RNA sequences from diverse sources. The consensus among virologists is that the base sequence of the novel coronavirus, designated SARS-CoV-2, was derived from a common ancestor of a bat coronavirus, represented by the strain RaTG13, isolated in Yunnan province in 2013. Into that ancestral genetic background, several recombination events have since occurred from other divergent bat-derived coronaviruses, resulting in localized discordance between the 2. One such event left SARS-CoV-2 with a receptor binding domain (RBD) capable of binding the human ACE-2 receptor lacking in RaTG13, and a 2nd event uniquely added to SARS-CoV-2 a site specific for furin, capable of efficient endoproteolytic cleavage and activation of the spike glycoprotein responsible for virus entry and cell fusion. This paper demonstrates by bioinformatic anal. that such recombinational events are facilitated by short oligonucleotide breakpoint sequences, similar to CAGAC, that direct recombination naturally to certain positions in the genome at the boundaries between blocks of RNA code and potentially RNA structure. This breakpoint sequence hypothesis provides a natural explanation for the biogenesis of SARS-CoV-2 over time and in the wild.
- 39Lytras, S., MacLean, O. A., and Robertson, D. L. (2020) The Sarbecovirus origin of SARS-CoV-2’s furin cleavage site, https://virological.org/t/the-sarbecovirus-origin-of-sars-cov-2-s-furin-cleavage-site/536.Google ScholarThere is no corresponding record for this reference.
- 40Yang, Y., Du, L., Liu, C., Wang, L., Ma, C., Tang, J., Baric, R. S., Jiang, S., and Li, F. (2014) Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. U. S. A. 111, 12516– 12521, DOI: 10.1073/pnas.1405889111Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlCju7rI&md5=2050deb7cfca13f5bb1c623c3df11172Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirusYang, Yang; Du, Lanying; Liu, Chang; Wang, Lili; Ma, Cuiqing; Tang, Jian; Baric, Ralph S.; Jiang, Shibo; Li, FangProceedings of the National Academy of Sciences of the United States of America (2014), 111 (34), 12516-12521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.
- 41Mcgrath, M. E. (1999) The Lysosomal Cysteine Proteases. Annu. Rev. Biophys. Biomol. Struct. 28, 181– 204, DOI: 10.1146/annurev.biophys.28.1.181Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXkt1erur4%253D&md5=fbdfd63ed7ff62e601720efe1e0db2caThe lysosomal cysteine proteasesMcGrath, Mary E.Annual Review of Biophysics and Biomolecular Structure (1999), 28 (), 181-204CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)A review with 112 refs. A significant no. of exciting papain-like cysteine protease structures have been detd. by crystallog. methods over the last several years. This trove of data allows for an anal. of the structural features that empower these mols. as they efficiently carry out their specialized tasks. Although the structure of the paradigm for the family, papain, has been known for 20 yr, recent efforts have reaped several structures of specialized mammalian enzymes. This review 1st covers the commonalities of architecture and purpose of the papain-like cysteine proteases. From that broad platform, each of the lysosomal enzymes for which there is an x-ray structure (or structures) is then examd. to gain an understanding of what structural features are used to customize specificity and activity. Structure-based design of inhibitors to control pathol. cysteine protease activity is also discussed.
- 42Laczkó, D., Hogan, M. J., Toulmin, S. A., Hicks, P., Lederer, K., Gaudette, B. T., Castraño, D., Amanat, F., Muramatsu, H., Oguin, T. H., Ojha, A., Zhang, L., Mu, Z., Parks, R., Manzoni, T. B., Roper, B., Strohmeier, S., Tombácz, I., Arwood, L., Nachbagauer, R., Karikó, K., Greenhouse, J., Pessaint, L., Porto, M., Putman-Taylor, T., Strasbaugh, A., Campbell, T. A., Lin, P. J., Tam, Y. K., Sempowski, G. D., Farzan, M., Choe, H., Saunders, K. O., Haynes, B. F., Andersen, H., Eisenlohr, L. C., Weissman, D., Krammer, F., Bates, P., Allman, D., Locci, M., and Pardi, N. (2020) A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity 53, 724– 732.E7, DOI: 10.1016/j.immuni.2020.07.019Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOhtb%252FE&md5=17ee76cb17463311cda626b83fcb5269A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in MiceLaczko, Dorottya; Hogan, Michael J.; Toulmin, Sushila A.; Hicks, Philip; Lederer, Katlyn; Gaudette, Brian T.; Castano, Diana; Amanat, Fatima; Muramatsu, Hiromi; Oguin, Thomas H., III; Ojha, Amrita; Zhang, Lizhou; Mu, Zekun; Parks, Robert; Manzoni, Tomaz B.; Roper, Brianne; Strohmeier, Shirin; Tombacz, Istvan; Arwood, Leslee; Nachbagauer, Raffael; Kariko, Katalin; Greenhouse, Jack; Pessaint, Laurent; Porto, Maciel; Putman-Taylor, Tammy; Strasbaugh, Amanda; Campbell, Tracey-Ann; Lin, Paulo J. C.; Tam, Ying K.; Sempowski, Gregory D.; Farzan, Michael; Choe, Hyeryun; Saunders, Kevin O.; Haynes, Barton F.; Andersen, Hanne; Eisenlohr, Laurence C.; Weissman, Drew; Krammer, Florian; Bates, Paul; Allman, David; Locci, Michela; Pardi, NorbertImmunity (2020), 53 (4), 724-732.e7CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality assocd. with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Addnl., we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.
- 43Cheng, Y.-W., Chao, T.-L., Li, C.-L., Chiu, M.-F., Kao, H.-C., Wang, S.-H., Pang, Y.-H., Lin, C.-H., Tsai, Y.-M., Lee, W.-H., Tao, M.-H., Ho, T.-C., Wu, P.-Y., Jang, L.-T., Chen, P.-J., Chang, S.-Y., and Yeh, S.-H. (2020) Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 33, 108254, DOI: 10.1016/j.celrep.2020.108254Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOhur7N&md5=38487fb54a3d9259e015b03850cdedf8Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic EffectsCheng, Ya-Wen; Chao, Tai-Ling; Li, Chiao-Ling; Chiu, Mu-Fan; Kao, Han-Chieh; Wang, Sheng-Han; Pang, Yu-Hao; Lin, Chih-Hui; Tsai, Ya-Min; Lee, Wen-Hau; Tao, Mi-Hua; Ho, Tung-Ching; Wu, Ping-Yi; Jang, Li-Ting; Chen, Pei-Jer; Chang, Sui-Yuan; Yeh, Shiou-HweiCell Reports (2020), 33 (2), 108254CODEN: CREED8; ISSN:2211-1247. (Cell Press)A review. Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, crit. for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus prodn. and cytopathic effects. Further anal. reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.
- 44Edridge, A. W. D., Kaczorowska, J., Hoste, A. C. R., Bakker, M., Klein, M., Loens, K., Jebbink, M. F., Matser, A., Kinsella, C. M., Rueda, P., Ieven, M., Goossens, H., Prins, M., Sastre, P., Deijs, M., and van der Hoek, L. (2020) Seasonal coronavirus protective immunity is short-lasting. Nat. Med. 26, 1691, DOI: 10.1038/s41591-020-1083-1Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVanurrL&md5=a5bd19cb9033ef4564acf7bb6a02f067Seasonal coronavirus protective immunity is short-lastingEdridge, Arthur W. D.; Kaczorowska, Joanna; Hoste, Alexis C. R.; Bakker, Margreet; Klein, Michelle; Loens, Katherine; Jebbink, Maarten F.; Matser, Amy; Kinsella, Cormac M.; Rueda, Paloma; Ieven, Margareta; Goossens, Herman; Prins, Maria; Sastre, Patricia; Deijs, Martin; van der Hoek, LiaNature Medicine (New York, NY, United States) (2020), 26 (11), 1691-1693CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)A key unsolved question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of acquired immunity. Insights from infections with the four seasonal human coronaviruses might reveal common characteristics applicable to all human coronaviruses. We monitored healthy individuals for more than 35 years and detd. that reinfection with the same seasonal coronavirus occurred frequently at 12 mo after infection.
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Abstract
Figure 1
Figure 1. Predicted structural model of the SARS-CoV and SARS-CoV-2 S proteins. (Inset) Magnification of the S1/S2 site with conserved R and S residues (red ribbon) and the unique four amino acid insertion P–R–R–A for SARS-CoV-2 (blue ribbon) is shown. The P’s denote the position of that amino acid from the S1/S2 cleavage site, with P1–P5 referring to the amino acids before the cleavage site and P1′ referring to the amino acids after the cleavage site.
Figure 2
Figure 2. Characterization of an MLVpp system. (A, B) MLVpp infectivity in Vero E6 and Calu-3 cells. Cells were infected with MLVpps exhibiting the SARS-CoV-2 S, SARS-CoV S, VSV G, or no envelope protein and assessed for luciferase activity. Error bars represent the standard error measurements of three biological replicates (n = 3). (C) Western blot analysis of SARS-CoV-2pp and SARS-CoVpp S content. S was detected using a rabbit antibody against the SARS-CoV-2 S2 region and cross reacts against the SARS-CoV S. MLV content was detected using a mouse antibody against MLV p30. The image was cropped from a singular Western blot.
Figure 3
Figure 3. Impact of dec-RVKR-CMK on MLVpps. + dec-RVKR-CMK refers to MLVpp produced in HEK293T treated with 75 μM dec-RVKR-CMK at the time of transfection. + furin refers to particles treated with 6 U of recombinant furin for 3 h at 37 °C. (A, B) ± dec-RVKR-CMK and ± furin SARS-CoV-2pp were used to infect Vero E6 and Calu-3 cells. Infectivity was normalized to the – dec-RVKR-CMK, – furin condition in each cell line. Note the change in y-axis scale between A and B. Error bars represent the standard error measurements of three biological replicates (n = 3). Statistical analysis was performed using an unpaired student’s t test. Not shown: there is a ns difference between the −dec-RVKR-CMK, ± furin conditions (black bars). *, P ≤ 0.05; **, P ≤ 0.01; ns, nonsignificant, P > 0.05. (C) Western blot analysis of SARS-CoV-2pp produced in ± dec-RVKR-CMK and treated with ± furin for S protein content to complement the infection conditions. S was detected using a rabbit antibody against the SARS-CoV-2 S2 region. MLV content was detected using a mouse antibody against MLV p30. Band intensities were normalized to the MLV p30 band intensity.
Figure 4
Figure 4. Use of specific furin (alpha1-PDX) and broad furin (dec-RVKR-CMK) inhibitors to investigate furin specificity. (A) Infectivity of particles produced in the presence of broad (dec-RVKR-CMK) and specific (alpha1-PDX) furin inhibitors. The inhibitor was applied at the indicated concentration to producer HEK293T cells. Particles were used to infect Vero E6 cells. Infectivity was normalized to the – condition. Error bars represent the standard error measurements of four biological replicates (n = 4). Statistical analysis was performed using an unpaired student’s t test compared to the – condition. *, P ≤ 0.05; ns, nonsignificant, P > 0.05. (B) Proteolytic cleavage assay of SARS-CoV-2 S1/S2 peptides to confirm alpha1-PDX and dec-RVKR-CMK inhibition specificity. Alpha1-PDX (2 μM), dec-RVKR-CMK (75 μM), or no inhibitor was incubated with either furin or trypsin recombinant protease and a fluorogenic peptide mimicking the SARS-CoV-2 S1/S2 site (TNSPRRARSVA). Results represent averages, and error bars represent the standard error measurement of three biological replicates (n = 3).
Figure 5
Figure 5. Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV S sequences were analyzed using the ProP (35) 1.0 and PiTou (34) 3.0 furin prediction algorithm. Bold scores indicate the S1/S2 sequence is predicted to be cleaved by furin. Red box highlights SARS-CoV and SARS-CoV-2 scores for reader’s convenience. Purple lines denote the position of the predicted S1/S2 cleavage site. Basic arginine (R) and lysine (K) residues are highlighted in blue. *For Bat-RmYN02, the sequence number was determined from the S alignment with SARS-CoV-2 S using Geneious. Sequences corresponding to the S1/S2 region of HCoV-HKU1 (AAT98580.1), SARS-CoV (AAT74874.1), SARS-CoV-2 (QHD43416.1), Bat-CoVRaTG13 (QHR63300.2), Bat-SL-CoVZC45 (AVP78031.1), Bat-SL-CoVZXC21 (AVP78042.1), MERS-CoV (AFS88936.1), BatCoV-HKU4 (YP_001039953.1), BatCoV-HKU5 (YP_001039962.1), BatCoV-PML (KC869678), Bat-CoV-HKU9 (YP_001039971), and Influenza A/Chicken/Hong Kong/822.1/01/H5N1 (AF509026.2) were obtained from GenBank. The sequence corresponding to the S1/S2 region of RmYN02 (EPI_ISL_412977) was obtained from GISAID.
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- 4Belouzard, S., Millet, J. K., Licitra, B. N., and Whittaker, G. R. (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4, 1011– 1033, DOI: 10.3390/v40610114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpvFyltrk%253D&md5=327223944a652c715b0a1550d0e4038aMechanisms of coronavirus cell entry mediated by the viral spike proteinBelouzard, Sandrine; Millet, Jean K.; Licitra, Beth N.; Whittaker, Gary R.Viruses (2012), 4 (), 1011-1033CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)A review. Coronaviruses are enveloped pos.-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes - A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
- 5White, J. M. and Whittaker, G. R. (2016) Fusion of Enveloped Viruses in Endosomes. Traffic 17, 593– 614, DOI: 10.1111/tra.123895https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlslWls70%253D&md5=915c967ab0f18e75e824f6cfc3de5cdfFusion of enveloped viruses in endosomesWhite, Judith M.; Whittaker, Gary R.Traffic (Oxford, United Kingdom) (2016), 17 (6), 593-614CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)A review. Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biol. in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. The authors end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid compn.) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion.
- 6Heald-Sargent, T. and Gallagher, T. (2012) Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence. Viruses 4, 557– 580, DOI: 10.3390/v40405576https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslOqsbk%253D&md5=68753d01793151eea573e5e90a853877Ready, set, fuse! the coronavirus spike protein and acquisition of fusion competenceHeald-Sargent, Taylor; Gallagher, TomViruses (2012), 4 (), 557-580CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)A review. Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies.
- 7Park, J. E., Li, K., Barlan, A., Fehr, A. R., Perlman, S., McCray, P. B., and Gallagher, T. (2016) Proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism. Proc. Natl. Acad. Sci. U. S. A. 113, 12262– 12267, DOI: 10.1073/pnas.16081471137https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1elur3F&md5=25764c2eb9d46c8ab09f70c461f8cb95Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropismPark, Jung-Eun; Li, Kun; Barlan, Arlene; Fehr, Anthony R.; Perlman, Stanley; McCray, Paul B. Jr.; Gallagher, TomProceedings of the National Academy of Sciences of the United States of America (2016), 113 (43), 12262-12267CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus-cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely det. how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.
- 8Shulla, A., Heald-Sargent, T., Subramanya, G., Zhao, J., Perlman, S., and Gallagher, T. (2011) A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry Downloaded from. J. Virol. 85, 873– 882, DOI: 10.1128/JVI.02062-108https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXisVOktLg%253D&md5=3dd5d16397b36bda4d781044694321bdA transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entryShulla, Ana; Heald-Sargent, Taylor; Subramanya, Gitanjali; Zhao, Jinchun; Perlman, Stanley; Gallagher, TomJournal of Virology (2011), 85 (2), 873-882CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunopptn. captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a sep. endoprotease operates as a portal for activation of SARS-CoV cell entry.
- 9Matsuyama, S., Nagata, N., Shirato, K., Kawase, M., Takeda, M., and Taguchi, F. (2010) Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. J. Virol. 84, 12658– 12664, DOI: 10.1128/JVI.01542-109https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlsVejtA%253D%253D&md5=01c4cb62576a1047d696d1712c68bdbaEfficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2Matsuyama, Shutoku; Nagata, Noriyo; Shirato, Kazuya; Kawase, Miyuki; Takeda, Makoto; Taguchi, FumihiroJournal of Virology (2010), 84 (24), 12658-12664CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, prodn. of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.
- 10Glowacka, I., Bertram, S., Müller, M. A., Allen, P., Soilleux, E., Pfefferle, S., Steffen, I., Tsegaye, T. S., He, Y., Gnirss, K., Niemeyer, D., Schneider, H., Drosten, C., and Pöhlmann, S. (2011) Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response. J. Virol. 85, 4122– 4134, DOI: 10.1128/JVI.02232-1010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MvjvF2ntA%253D%253D&md5=94e4268b223e658c2b592d2e1cf19839Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune responseGlowacka Ilona; Bertram Stephanie; Muller Marcel A; Allen Paul; Soilleux Elizabeth; Pfefferle Susanne; Steffen Imke; Tsegaye Theodros Solomon; He Yuxian; Gnirss Kerstin; Niemeyer Daniela; Schneider Heike; Drosten Christian; Pohlmann StefanJournal of virology (2011), 85 (9), 4122-34 ISSN:.The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.
- 11Simmons, G., Gosalia, D. N., Rennekamp, A. J., Reeves, J. D., Diamond, S. L., and Bates, P. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc. Natl. Acad. Sci. U. S. A. 102, 11876– 11881, DOI: 10.1073/pnas.050557710211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsFGgurw%253D&md5=99f26bc819463a5377187542db9bdfe5Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entrySimmons, Graham; Gosalia, Dhaval N.; Rennekamp, Andrew J.; Reeves, Jacqueline D.; Diamond, Scott L.; Bates, PaulProceedings of the National Academy of Sciences of the United States of America (2005), 102 (33), 11876-11881CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currently no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) glycoprotein, and infection is sensitive to lysosomotropic agents that perturb endosomal pH. We demonstrate here that the lysosomotropic-agent-mediated block to SARS-CoV infection is overcome by protease treatment of target-cell-assocd. virus. In addn., SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. A cell-free membrane-fusion system demonstrates that engagement of receptor followed by proteolysis is required for SARS-CoV membrane fusion and indicates that cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These results suggest that SARS-CoV infection results from a unique, three-step process: receptor binding and induced conformational changes in S glycoprotein followed by cathepsin L proteolysis within endosomes. The requirement for cathepsin L proteolysis identifies a previously uncharacterized class of inhibitor for SARS-CoV infection.
- 12Millet, J. K. and Whittaker, G. R. (2014) Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein. Proc. Natl. Acad. Sci. U. S. A. 111, 15214– 15219, DOI: 10.1073/pnas.140708711112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1yhtb7E&md5=21bea9924d802dd56de1dac9d38bd509Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike proteinMillet, Jean Kaoru; Whittaker, Gary R.Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (42), 15214-15219CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2' position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2' site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus.
- 13Reinke, L. M., Spiegel, M., Plegge, T., Hartleib, A., Nehlmeier, I., Gierer, S., Hoffmann, M., Hofmann-Winkler, H., Winkler, M., and Pöhlmann, S. (2017) Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS One 12 (6), e0179177 DOI: 10.1371/journal.pone.017917713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFynt7Y%253D&md5=b4e700c4fed413eb6f5b4358b1cf010dDifferent residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Poehlmann, StefanPLoS One (2017), 12 (6), e0179177/1-e0179177/15CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.
- 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.abb250714https://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.
- 15Jaimes, J. A., André, N. M., Chappie, J. S., Millet, J. K., and Whittaker, G. R. (2020) Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J. Mol. Biol. 432, 3309– 3325, DOI: 10.1016/j.jmb.2020.04.00915https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnvFeju78%253D&md5=887ae39cca30bb371f36d7980c4c76d1Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation LoopJaimes, Javier A.; Andre, Nicole M.; Chappie, Joshua S.; Millet, Jean K.; Whittaker, Gary R.Journal of Molecular Biology (2020), 432 (10), 3309-3325CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) originally arose as part of a major outbreak of respiratory disease centered on Hubei province, China. It is now a global pandemic and is a major public health concern. Taxonomically, SARS-CoV-2 was shown to be a Betacoronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2). Subsequently, betacoronaviruses from pangolins were identified as close relatives to SARS-CoV-2. Here, we perform structural modeling of the SARS-CoV-2 spike glycoprotein. Our data provide support for the similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, we identify an extended structural loop contg. basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains. We suggest this loop confers fusion activation and entry properties more in line with betacoronaviruses in lineages A and C, and be a key component in the evolution of SARS-CoV-2 with this structural loop affecting virus stability and transmission.
- 16Henrich, S., Lindberg, I., Bode, W., and Than, M. E. (2005) Proprotein convertase models based on the crystal structures of furin and kexin: Explanation of their specificity. J. Mol. Biol. 345, 211– 227, DOI: 10.1016/j.jmb.2004.10.05016https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2cnjs1Sgtw%253D%253D&md5=667d12e3a21b22016d05d9108ff2bd33Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificityHenrich Stefan; Lindberg Iris; Bode Wolfram; Than Manuel EJournal of molecular biology (2005), 345 (2), 211-27 ISSN:0022-2836.In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.
- 17Henrich, S., Cameron, A., Bourenkov, G. P., Kiefersauer, R., Huber, R., Lindberg, I., Bode, W., and Than, M. E. (2003) The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat. Struct. Mol. Biol. 10, 520– 526, DOI: 10.1038/nsb941There is no corresponding record for this reference.
- 18Tian, S., Huang, Q., Fang, Y., and Wu, J. (2011) FurinDB: A Database of 20-residue furin cleavage site motifs, substrates and their associated drugs. Int. J. Mol. Sci. 12, 1060– 1065, DOI: 10.3390/ijms1202106018https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXitlSntb8%253D&md5=6baad48472fc42c9b938183c34102fffFurinDB: a database of 20-residue furin cleavage site motifs, substrates and their associated drugsTian, Sun; Huang, Qingsheng; Fang, Ying; Wu, JianhuaInternational Journal of Molecular Sciences (2011), 12 (), 1060-1065CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)FurinDB (freely available online) is a database of furin substrates. This database includes exptl. verified furin cleavage sites, substrates, species, exptl. methods, original publications of expts. and assocd. drugs targeting furin substrates. The current database release contains 126 furin cleavage sites from three species: mammals, bacteria and viruses. A main feature of this database is that all furin cleavage sites are recorded as a 20-residue motif, including one core region (eight amino acids, P6-P2') and two flanking solvent accessible regions (eight amino acids, P7-P14, and four amino acids, P3'-P6'), that represent our current understanding of the mol. biol. of furin cleavage. This database is important for understanding the mol. evolution and relationships between sequence motifs, 3D structures, cellular functions and phys. properties required by furin for cleavage, and for elucidating the mol. mechanisms and the progression of furin cleavage assocd. human diseases, including pathogenic infections, neurol. disorders, tumorigenesis, tumor invasion, angiogenesis, and metastasis. FurinDB database will be a solid addn. to the publicly available infrastructure for scientists in the field of mol. biol.
- 19Abrami, L., Fivaz, M., Decroly, E., Seidah, N. G., Jean, F., Thomas, G., Leppla, S. H., Buckley, J. T., and Van Der Goot, F. G. (1998) The pore-forming toxin proaerolysin is activated by furin. J. Biol. Chem. 273, 32656– 32661, DOI: 10.1074/jbc.273.49.3265619https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXnvFOntb4%253D&md5=3aa5f2f2855acb2901c6890841478e68The pore-forming toxin proaerolysin is activated by furinAbrami, Laurence; Fivaz, Marc; Decroly, Etienne; Seidah, Nabil G.; Jean, Francois; Thomas, Gary; Leppla, Stephen H.; Buckley, J. Thomas; Van Der Goot, F. GisouJournal of Biological Chemistry (1998), 273 (49), 32656-32661CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor α1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.
- 20Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, T. S., Herrler, G., Wu, N. H., Nitsche, A., Müller, M. A., Drosten, C., and Pöhlmann, S. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280.E8, DOI: 10.1016/j.cell.2020.02.05220https://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.
- 21Walls, 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, DOI: 10.1016/j.cell.2020.02.05821https://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.
- 22Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J., and Qian, Z. (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620, DOI: 10.1038/s41467-020-15562-922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFyjt78%253D&md5=6b0b1ef5a68f4a35da4aabecb0f99544Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoVOu, Xiuyuan; Liu, Yan; Lei, Xiaobo; Li, Pei; Mi, Dan; Ren, Lili; Guo, Li; Guo, Ruixuan; Chen, Ting; Hu, Jiaxin; Xiang, Zichun; Mu, Zhixia; Chen, Xing; Chen, Jieyong; Hu, Keping; Jin, Qi; Wang, Jianwei; Qian, ZhaohuiNature Communications (2020), 11 (1), 1620CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
- 23Hoffmann, M., Kleine-Weber, H., and Pöhlmann, S. (2020) A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol. Cell 78, 779– 784.E5, DOI: 10.1016/j.molcel.2020.04.02223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovVCgs7o%253D&md5=18c626bd22d3d9c280d8b5e19c116db8A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung CellsHoffmann, Markus; Kleine-Weber, Hannah; Poehlmann, StefanMolecular Cell (2020), 78 (4), 779-784.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site contg. multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
- 24Bestle, D., Heindl, M. R., Limburg, H., Van Lam van, T., Pilgram, O., Moulton, H., Stein, D. A., Hardes, K., Eickmann, M., Dolnik, O., Rohde, C., Klenk, H. D., Garten, W., Steinmetzer, T., and Böttcher-Friebertshäuser, E. (2020) TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci. alliance 3, e202000786, DOI: 10.26508/lsa.202000786There is no corresponding record for this reference.
- 25Millet, 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. 2019, e59010, DOI: 10.3791/59010There is no corresponding record for this reference.
- 26Straus, M. R., Tang, T., Lai, A. L., Flegel, A., Bidon, M., Freed, J. H., Daniel, S., and Whittaker, G. R. (2020) Ca 2+ ions promote fusion of Middle East respiratory syndrome coronavirus with host cells and increase infectivity. J. Virol. 94, 426– 446, DOI: 10.1128/JVI.00426-2026https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVKhurrF&md5=d8475cfbe72278344793d2ff2aff99abCa2+ ions promote fusion of middle east respiratory syndrome coronavirus with host cells and increase infectivityStraus, Marco R.; Tang, Tiffany; Lai, Alex L.; Flegel, Annkatrin; Bidon, Miya; Freed, Jack H.; Daniel, Susan; Whittaker, Gary R.Journal of Virology (2020), 94 (13), e00426-20CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Fusion with, and subsequent entry into, the host cell is one of the crit. steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homol., and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approx. 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent ESR (ESR) expts. revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approx. 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.
- 27Tang, T., Bidon, M., Jaimes, J. A., Whittaker, G. R., and Daniel, S. (2020) Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res. 178, 104792, DOI: 10.1016/j.antiviral.2020.10479227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsFalt7w%253D&md5=08a629b31ad760c3bf19c2175abe2363Coronavirus membrane fusion mechanism offers a potential target for antiviral developmentTang, Tiffany; Bidon, Miya; Jaimes, Javier A.; Whittaker, Gary R.; Daniel, SusanAntiviral Research (2020), 178 (), 104792CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)A review. The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapies against the causative agent, SARS-CoV-2, and also against other pathogenic coronaviruses (CoV) that have emerged in the past or might appear in future. Researchers are therefore focusing on steps in the CoV replication cycle that may be vulnerable to inhibition by broad-spectrum or specific antiviral agents. The conserved nature of the fusion domain and mechanism across the CoV family make it a valuable target to elucidate and develop pan-CoV therapeutics. In this article, we review the role of the CoV spike protein in mediating fusion of the viral and host cell membranes, summarizing the results of research on SARS-CoV, MERS-CoV, and recent peer-reviewed studies of SARS-CoV-2, and suggest that the fusion mechanism be investigated as a potential antiviral target. We also provide a supplemental file contg. background information on the biol., epidemiol., and clin. features of all human-infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.
- 28Hyseni, I., Molesti, E., Benincasa, L., Piu, P., Casa, E., Temperton, N. J., Manenti, A., and Montomoli, E. (2020) Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays. Viruses 12, 1011, DOI: 10.3390/v12091011There is no corresponding record for this reference.
- 29Matsuyama, S., Shirato, K., Kawase, M., Terada, Y., Kawachi, K., Fukushi, S., and Kamitani, W. (2018) Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells. J. Virol. 92, e00683-18, DOI: 10.1128/JVI.00683-18There is no corresponding record for this reference.
- 30Wrobel, A. G., Benton, D. J., Xu, P., Roustan, C., Martin, S. R., Rosenthal, P. B., Skehel, J. J., and Gamblin, S. J. (2020) SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 27, 763– 767, DOI: 10.1038/s41594-020-0468-730https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlGlurfM&md5=75e5b3c971677ffd82d98090b57255ecSARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effectsWrobel, Antoni G.; Benton, Donald J.; Xu, Pengqi; Roustan, Chloe; Martin, Stephen R.; Rosenthal, Peter B.; Skehel, John J.; Gamblin, Steven J.Nature Structural & Molecular Biology (2020), 27 (8), 763-767CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. We investigate the relation of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We detd. cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochem. characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and ∼1000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
- 31Jean, F., Stella, K., Thomas, L., Liu, G., Xiang, Y., Reason, A. J., and Thomas, G. (1998) α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent. Proc. Natl. Acad. Sci. U. S. A. 95, 7293– 7298, DOI: 10.1073/pnas.95.13.729331https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXktFChtr0%253D&md5=af8752ea7d2cac8404c0b858a9f7d6a4α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agentJean, Francois; Stella, Kori; Thomas, Laurel; Liu, Gseping; Xiang, Yang; Reason, Andrew J.; Thomas, GaryProceedings of the National Academy of Sciences of the United States of America (1998), 95 (13), 7293-7298CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The important role of furin in the proteolytic activation of many pathogenic mols. has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochem. anal. of the specificity of a bacterially expressed His-and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin mols. and functions as a suicide substrate inhibitor. Once bound to furin's active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center-Arg355-Ile-Pro-Arg358- or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small mol. furin inhibitors.
- 32Jaimes, J. A., Millet, J. K., and Whittaker, G. R. (2020) Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site. iScience 23, 101212, DOI: 10.1016/j.isci.2020.10121232https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFahur7I&md5=fb4f2beee3529e458165e526f75377f8Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 SiteJaimes, Javier A.; Millet, Jean K.; Whittaker, Gary R.iScience (2020), 23 (6), 101212CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochem. evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.
- 33Coutard, B., Valle, C., de Lamballerie, X., Canard, B., Seidah, N. G., and Decroly, E. (2020) The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res. 176, 104742, DOI: 10.1016/j.antiviral.2020.10474233https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjtFertr0%253D&md5=144e90f8ac4dc1a517c899b10c4a7a98The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same cladeCoutard, B.; Valle, C.; de Lamballerie, X.; Canard, B.; Seidah, N. G.; Decroly, E.Antiviral Research (2020), 176 (), 104742CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.
- 34Tian, S., Huajun, W., and Wu, J. (2012) Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseases. Sci. Rep. 2, 261, DOI: 10.1038/srep0026134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC383ltl2ktQ%253D%253D&md5=e9467e011fabed34fe6da3a913697d35Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseasesTian Sun; Huajun Wang; Wu JianhuaScientific reports (2012), 2 (), 261 ISSN:.Furin cleaves diverse types of protein precursors in the secretory pathway. The substrates for furin cleavage possess a specific 20-residue recognition sequence motif. In this report, based on the functional characterisation of the 20-residue sequence motif, we developed a furin cleavage site prediction tool, PiTou, using a hybrid method composed of a hidden Markov model and biological knowledge-based cumulative probability score functions. PiTou can accurately predict the presence and location of furin cleavage sites in protein sequences with high sensitivity (96.9%) and high specificity (97.3%). PiTou's prediction scores are biological meaningful and reflect binding strength and solvent accessibility of furin substrates. A prediction result is interpreted within cellular contexts: subcellular localisation, cellular function and interference by other dynamic protein modifications. Combining next-generation sequencing, PiTou can help with elucidating the molecular mechanism of furin cleavage-associated human diseases. PiTou has been made freely available at the associated website.
- 35Duckert, P., Brunak, S., and Blom, N. (2004) Prediction of proprotein convertase cleavage sites. Protein Eng., Des. Sel. 17, 107– 112, DOI: 10.1093/protein/gzh01335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlKgsbw%253D&md5=042a34ce1e8a987079be99bf0f60f104Prediction of proprotein convertase cleavage sitesDuckert, Peter; Brunak, Soren; Blom, NikolajProtein Engineering, Design & Selection (2004), 17 (1), 107-112CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)Many secretory proteins and peptides are synthesized as inactive precursors that, in addn. to signal peptide cleavage, undergo post-translational processing to become biol. active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created addnl. focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on exptl. results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network.
- 36Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C., and Garry, R. F. (2020) The proximal origin of SARS-CoV-2. Nat. Med. 26, 450– 452, DOI: 10.1038/s41591-020-0820-936https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXltFCjtbY%253D&md5=3489259c33e29365c0c1cf7fc5613407The proximal origin of SARS-CoV-2Andersen, Kristian G.; Rambaut, Andrew; Lipkin, W. Ian; Holmes, Edward C.; Garry, Robert F.Nature Medicine (New York, NY, United States) (2020), 26 (4), 450-452CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)There is no expanded citation for this reference.
- 37Boni, M. F., Lemey, P., Jiang, X., Lam, T. T. Y., Perry, B. W., Castoe, T. A., Rambaut, A., and Robertson, D. L. (2020) Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. Nat. Microbiol. 5, 1408– 1417, DOI: 10.1038/s41564-020-0771-437https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVyjt7vP&md5=d9469eec71c82371598422c09bb5534bEvolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemicBoni, Maciej F.; Lemey, Philippe; Jiang, Xiaowei; Lam, Tommy Tsan-Yuk; Perry, Blair W.; Castoe, Todd A.; Rambaut, Andrew; Robertson, David L.Nature Microbiology (2020), 5 (11), 1408-1417CODEN: NMAICH; ISSN:2058-5276. (Nature Research)Abstr.: There are outstanding evolutionary questions on the recent emergence of human coronavirus SARS-CoV-2 including the role of reservoir species, the role of recombination and its time of divergence from animal viruses. We find that the sarbecoviruses-the viral subgenus contg. SARS-CoV and SARS-CoV-2-undergo frequent recombination and exhibit spatially structured genetic diversity on a regional scale in China. SARS-CoV-2 itself is not a recombinant of any sarbecoviruses detected to date, and its receptor-binding motif, important for specificity to human ACE2 receptors, appears to be an ancestral trait shared with bat viruses and not one acquired recently via recombination. To employ phylogenetic dating methods, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Bayesian evolutionary rate and divergence date ests. were shown to be consistent for these three approaches and for two different prior specifications of evolutionary rates based on HCoV-OC43 and MERS-CoV. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estd. as 1948 (95% highest posterior d. (HPD): 1879-1999), 1969 (95% HPD: 1930-2000) and 1982 (95% HPD: 1948-2009), indicating that the lineage giving rise to SARS-CoV-2 has been circulating unnoticed in bats for decades.
- 38Gallaher, W. R. (2020) A palindromic RNA sequence as a common breakpoint contributor to copy-choice recombination in SARS-COV-2. Arch. Virol. 165, 2341– 2348, DOI: 10.1007/s00705-020-04750-z38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFShu7bN&md5=29efb730adb00258d37baeea1ebf0a50A palindromic RNA sequence as a common breakpoint contributor to copy-choice recombination in SARS-COV-2Gallaher, William R.Archives of Virology (2020), 165 (10), 2341-2348CODEN: ARVIDF; ISSN:0304-8608. (Springer-Verlag GmbH)Much remains unknown concerning the origin of the novel pandemic coronavirus that has raged across the globe since emerging in Wuhan of Hubei province, near the center of the People's Republic of China, in Dec. of 2019. All current members of the family Coronaviridae have arisen by a combination of incremental adaptive mutations, against the backdrop of many recombinational events throughout the past, rendering each a unique mosaic of RNA sequences from diverse sources. The consensus among virologists is that the base sequence of the novel coronavirus, designated SARS-CoV-2, was derived from a common ancestor of a bat coronavirus, represented by the strain RaTG13, isolated in Yunnan province in 2013. Into that ancestral genetic background, several recombination events have since occurred from other divergent bat-derived coronaviruses, resulting in localized discordance between the 2. One such event left SARS-CoV-2 with a receptor binding domain (RBD) capable of binding the human ACE-2 receptor lacking in RaTG13, and a 2nd event uniquely added to SARS-CoV-2 a site specific for furin, capable of efficient endoproteolytic cleavage and activation of the spike glycoprotein responsible for virus entry and cell fusion. This paper demonstrates by bioinformatic anal. that such recombinational events are facilitated by short oligonucleotide breakpoint sequences, similar to CAGAC, that direct recombination naturally to certain positions in the genome at the boundaries between blocks of RNA code and potentially RNA structure. This breakpoint sequence hypothesis provides a natural explanation for the biogenesis of SARS-CoV-2 over time and in the wild.
- 39Lytras, S., MacLean, O. A., and Robertson, D. L. (2020) The Sarbecovirus origin of SARS-CoV-2’s furin cleavage site, https://virological.org/t/the-sarbecovirus-origin-of-sars-cov-2-s-furin-cleavage-site/536.There is no corresponding record for this reference.
- 40Yang, Y., Du, L., Liu, C., Wang, L., Ma, C., Tang, J., Baric, R. S., Jiang, S., and Li, F. (2014) Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. U. S. A. 111, 12516– 12521, DOI: 10.1073/pnas.140588911140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlCju7rI&md5=2050deb7cfca13f5bb1c623c3df11172Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirusYang, Yang; Du, Lanying; Liu, Chang; Wang, Lili; Ma, Cuiqing; Tang, Jian; Baric, Ralph S.; Jiang, Shibo; Li, FangProceedings of the National Academy of Sciences of the United States of America (2014), 111 (34), 12516-12521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.
- 41Mcgrath, M. E. (1999) The Lysosomal Cysteine Proteases. Annu. Rev. Biophys. Biomol. Struct. 28, 181– 204, DOI: 10.1146/annurev.biophys.28.1.18141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXkt1erur4%253D&md5=fbdfd63ed7ff62e601720efe1e0db2caThe lysosomal cysteine proteasesMcGrath, Mary E.Annual Review of Biophysics and Biomolecular Structure (1999), 28 (), 181-204CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)A review with 112 refs. A significant no. of exciting papain-like cysteine protease structures have been detd. by crystallog. methods over the last several years. This trove of data allows for an anal. of the structural features that empower these mols. as they efficiently carry out their specialized tasks. Although the structure of the paradigm for the family, papain, has been known for 20 yr, recent efforts have reaped several structures of specialized mammalian enzymes. This review 1st covers the commonalities of architecture and purpose of the papain-like cysteine proteases. From that broad platform, each of the lysosomal enzymes for which there is an x-ray structure (or structures) is then examd. to gain an understanding of what structural features are used to customize specificity and activity. Structure-based design of inhibitors to control pathol. cysteine protease activity is also discussed.
- 42Laczkó, D., Hogan, M. J., Toulmin, S. A., Hicks, P., Lederer, K., Gaudette, B. T., Castraño, D., Amanat, F., Muramatsu, H., Oguin, T. H., Ojha, A., Zhang, L., Mu, Z., Parks, R., Manzoni, T. B., Roper, B., Strohmeier, S., Tombácz, I., Arwood, L., Nachbagauer, R., Karikó, K., Greenhouse, J., Pessaint, L., Porto, M., Putman-Taylor, T., Strasbaugh, A., Campbell, T. A., Lin, P. J., Tam, Y. K., Sempowski, G. D., Farzan, M., Choe, H., Saunders, K. O., Haynes, B. F., Andersen, H., Eisenlohr, L. C., Weissman, D., Krammer, F., Bates, P., Allman, D., Locci, M., and Pardi, N. (2020) A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity 53, 724– 732.E7, DOI: 10.1016/j.immuni.2020.07.01942https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOhtb%252FE&md5=17ee76cb17463311cda626b83fcb5269A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in MiceLaczko, Dorottya; Hogan, Michael J.; Toulmin, Sushila A.; Hicks, Philip; Lederer, Katlyn; Gaudette, Brian T.; Castano, Diana; Amanat, Fatima; Muramatsu, Hiromi; Oguin, Thomas H., III; Ojha, Amrita; Zhang, Lizhou; Mu, Zekun; Parks, Robert; Manzoni, Tomaz B.; Roper, Brianne; Strohmeier, Shirin; Tombacz, Istvan; Arwood, Leslee; Nachbagauer, Raffael; Kariko, Katalin; Greenhouse, Jack; Pessaint, Laurent; Porto, Maciel; Putman-Taylor, Tammy; Strasbaugh, Amanda; Campbell, Tracey-Ann; Lin, Paulo J. C.; Tam, Ying K.; Sempowski, Gregory D.; Farzan, Michael; Choe, Hyeryun; Saunders, Kevin O.; Haynes, Barton F.; Andersen, Hanne; Eisenlohr, Laurence C.; Weissman, Drew; Krammer, Florian; Bates, Paul; Allman, David; Locci, Michela; Pardi, NorbertImmunity (2020), 53 (4), 724-732.e7CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality assocd. with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Addnl., we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.
- 43Cheng, Y.-W., Chao, T.-L., Li, C.-L., Chiu, M.-F., Kao, H.-C., Wang, S.-H., Pang, Y.-H., Lin, C.-H., Tsai, Y.-M., Lee, W.-H., Tao, M.-H., Ho, T.-C., Wu, P.-Y., Jang, L.-T., Chen, P.-J., Chang, S.-Y., and Yeh, S.-H. (2020) Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 33, 108254, DOI: 10.1016/j.celrep.2020.10825443https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOhur7N&md5=38487fb54a3d9259e015b03850cdedf8Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic EffectsCheng, Ya-Wen; Chao, Tai-Ling; Li, Chiao-Ling; Chiu, Mu-Fan; Kao, Han-Chieh; Wang, Sheng-Han; Pang, Yu-Hao; Lin, Chih-Hui; Tsai, Ya-Min; Lee, Wen-Hau; Tao, Mi-Hua; Ho, Tung-Ching; Wu, Ping-Yi; Jang, Li-Ting; Chen, Pei-Jer; Chang, Sui-Yuan; Yeh, Shiou-HweiCell Reports (2020), 33 (2), 108254CODEN: CREED8; ISSN:2211-1247. (Cell Press)A review. Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, crit. for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus prodn. and cytopathic effects. Further anal. reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.
- 44Edridge, A. W. D., Kaczorowska, J., Hoste, A. C. R., Bakker, M., Klein, M., Loens, K., Jebbink, M. F., Matser, A., Kinsella, C. M., Rueda, P., Ieven, M., Goossens, H., Prins, M., Sastre, P., Deijs, M., and van der Hoek, L. (2020) Seasonal coronavirus protective immunity is short-lasting. Nat. Med. 26, 1691, DOI: 10.1038/s41591-020-1083-144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVanurrL&md5=a5bd19cb9033ef4564acf7bb6a02f067Seasonal coronavirus protective immunity is short-lastingEdridge, Arthur W. D.; Kaczorowska, Joanna; Hoste, Alexis C. R.; Bakker, Margreet; Klein, Michelle; Loens, Katherine; Jebbink, Maarten F.; Matser, Amy; Kinsella, Cormac M.; Rueda, Paloma; Ieven, Margareta; Goossens, Herman; Prins, Maria; Sastre, Patricia; Deijs, Martin; van der Hoek, LiaNature Medicine (New York, NY, United States) (2020), 26 (11), 1691-1693CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)A key unsolved question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of acquired immunity. Insights from infections with the four seasonal human coronaviruses might reveal common characteristics applicable to all human coronaviruses. We monitored healthy individuals for more than 35 years and detd. that reinfection with the same seasonal coronavirus occurred frequently at 12 mo after infection.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00701.
Impact of the dec-RVKR-CMK inhibitor on SARS-CoVpp production, which lacks the furin S1/S2 site (PDF)
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