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Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and EntryClick to copy article linkArticle link copied!
- Mason R. FirpoMason R. FirpoDepartment of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United StatesMore by Mason R. Firpo
- Vincent MastrodomenicoVincent MastrodomenicoDepartment of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United StatesMore by Vincent Mastrodomenico
- Grant M. HawkinsGrant M. HawkinsDepartment of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United StatesMore by Grant M. Hawkins
- Matthieu ProtMatthieu ProtG5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris 75015, FranceMore by Matthieu Prot
- Laura LevillayerLaura LevillayerFunctional Genetics of Infectious Diseases Unit, Institut Pasteur, Paris 75015, FranceMore by Laura Levillayer
- Tom GallagherTom GallagherDepartment of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United StatesMore by Tom Gallagher
- Etienne Simon-LoriereEtienne Simon-LoriereG5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris 75015, FranceMore by Etienne Simon-Loriere
- Bryan C. Mounce*Bryan C. Mounce*Phone: 708 216 3358. Email: [email protected]Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United StatesMore by Bryan C. Mounce
Abstract
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Coronaviruses first garnered widespread attention in 2002 when the severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from bats in China and rapidly spread in human populations. Since then, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged and still actively infects humans. The recent SARS-CoV-2 outbreak and the resulting disease (coronavirus disease 2019, COVID19) have rapidly and catastrophically spread and highlighted significant limitations to our ability to control and treat infection. Thus, a basic understanding of entry and replication mechanisms of coronaviruses is necessary to rationally evaluate potential antivirals. Here, we show that polyamines, small metabolites synthesized in human cells, facilitate coronavirus replication and the depletion of polyamines with FDA-approved molecules significantly reduces coronavirus replication. We find that diverse coronaviruses, including endemic and epidemic coronaviruses, exhibit reduced attachment and entry into polyamine-depleted cells. We further demonstrate that several molecules targeting the polyamine biosynthetic pathway are antiviral in vitro. In sum, our data suggest that polyamines are critical to coronavirus replication and represent a highly promising drug target in the current and any future coronavirus outbreaks.
Copyright © 2020 American Chemical Society
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This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
Coronaviruses are significant human pathogens with significant epidemic potential. Recurring coronavirus outbreaks wreak havoc on human health and activity, as demonstrated by the original severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak of 2002, the Middle East respiratory syndrome coronavirus (MERS-CoV) of 2011 to present day, (1) and the newly emergent SARS-CoV-2 of 2019 to present. (2) Each of these viruses emerged from zoonotic sources and were rapidly transmitted through human populations, requiring urgent attention due to the number of infections, combined with mortality rates of up to 35% in the case of MERS-CoV. (3) In addition to their epidemic potential, coronaviruses are common human pathogens, with several endemic coronaviruses causing recurrent infections with generally mild symptoms. (4,5) Treatments for SARS-CoV-2, as well as other coronaviruses, include remdesivir, a base analog that exhibited promising clinical efficacy. (6,7) A dearth of additional antivirals is available to treat or prevent infections. The omnipresence, unpredictable epidemic potential, and significant impact on human health make the investigation into potential antiviral compounds essential to battle current and future coronavirus outbreaks.
Polyamines are small aliphatic metabolites synthesized by mammalian cells to support cellular processes such as cell cycling, transcription, and translation. (8−10) The polyamines putrescine, spermidine, and spermine (Figure 1A) are synthesized from an ornithine precursor through the enzyme ODC1. Several specific and potent inhibitors of the polyamine biosynthetic pathway have been developed and are FDA approved. The ODC1 inhibitor difluoromethylornithine (DFMO) is approved for the treatment of trypanosomiasis. (11) In fact, DFMO exhibits low toxicity and mild side effects. (12) Topical DFMO has also been developed and is sold as Vaniqa to reduce hair growth. (13) Viruses rely on polyamines for varied stages in replication, including polymerase activity, (14) cell binding, (15) and viral protein translation. (15,16) Polyamine depletion via DFMO effectively reduces the replication of diverse viruses in vitro and in vivo with minimal cytotoxicity. (17,18) Prior work demonstrated that MERS-CoV was sensitive to the polyamine inhibitor DFMO in vitro at a dose of 500 μM, (19) suggesting that polyamines function in coronavirus replication. Whether and how polyamines function in the replication of other coronaviruses remain to be investigated.
Figure 1
Figure 1. Polyamine synthesis inhibitor DFMO limits coronavirus replication. (A) Schematic of the polyamine pathway. The polyamines putrescine, spermidine, and spermine are synthesized from ornithine via ornithine decarboxylase (ODC1), which is inhibited by difluoromethylornithine (DFMO). (B) Vero-E6 cells treated with increasing doses of DFMO were infected with SARS-CoV-2 at an MOI of 0.1 for 24 h, and viral genomes were measured and converted to titer equivalents via a standard curve. (C) Vero-E6 cells were treated with escalating DFMO doses and viability and (D) polyamine levels were measured. (E) BHK-R cells were treated with DFMO as indicated and infected with MHV at an MOI of 0.01 for 48 h. (F) Cellular viability levels were measured and (G) polyamine depletion was confirmed by chromatography. (H) BHK-R cells were treated with 1 mM DFMO and infected with MHV at an MOI of 0.01. Viral titers were determined after several rounds of replication. (I) MHV genomes from samples in (E) were quantified by qRT-PCR and compared to viral titers to quantify the relative number of genomes to PFU (J). (K) HeLa-R cells were treated with escalating doses of DFMO and infected with MHV at an MOI of 0.01 for 48 h. (L) HeLa-R cells exposed to DFMO for 4 days were analyzed for viability. (M) Depletion of polyamines was confirmed by chromatography. *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
The study of human epidemic coronaviruses is limited due to requisite biosafety protocols. Thus, murine model systems have been developed to study coronavirus replication and pathogenesis with significantly reduced risk. Murine hepatitis virus (MHV) is a common model system for coronavirus infection. MHV, a β-coronavirus, is related to the human epidemic coronaviruses, though it is safe to work with in the laboratory. The MHV model is well characterized, (20,21) and combined with its ease of use, it provides a significant opportunity to study coronavirus infection.
Using the MHV model system, we investigated a role for polyamines in coronavirus infection. We observe that polyamine depletion via DFMO limits virus replication and that polyamines support virus infection at the stage of attachment: polyamine depletion reduces virus binding to susceptible cells. We extend this phenotype to the human endemic coronavirus NL63 as well as the pandemic SARS-CoV-2. Additionally, we demonstrate that several molecules targeting the polyamine pathway, including FDA-approved pharmaceuticals, exhibit significant antiviral activity. Together, our data highlight a role for polyamines in coronavirus replication and suggest that targeting polyamines may be a viable preventative or treatment option for coronavirus infection.
Results
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Polyamine Depletion Limits Coronavirus Replication
To determine if coronaviruses are susceptible to polyamine depletion, we treated Vero-E6 cells with doses of DFMO ranging from 750 μM to 5 mM for 4 days prior to infection with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 plaque forming units (pfu) per cell. Released viral genomes were then measured at 24 h post-infection (hpi) and converted to virus equivalents using a standard curve comparing known infectious titers and genomes. We observed that DFMO significantly reduced SARS-CoV-2 genomes, greater than 50-fold at 5 mM, with an IC50 (50% inhibitory concentration) value of 230 μM (Figure 1B). To confirm that DFMO was not cytotoxic, we measured the viability of Vero-E6 cells treated with escalating doses of DFMO and calculated a CC50 (50% cytotoxic concentration) value of 11 mM (Figure 1C; summarized in Table 1). We further confirmed that DFMO treatment at these concentrations successfully depleted polyamines via thin layer chromatography (TLC; Figure 1D).
Table 1. Properties of the Compounds Used in These Studies
| inhibitor | virus | cells | IC50 (μM) | CC50 (mM) | SI |
|---|---|---|---|---|---|
| DFMO | MHV-A59 | BHK-R | 380 | 3.8 | 10 |
| HeLa-R | 7.0 | 16 | 2300 | ||
| DENSpm | HeLa-R | 1.6 | 35 | 7100 | |
| CPX | BHK-R | 2.2 | 76 | 35000 | |
| DEF | BHK-R | 54 | 60 | 1100 | |
| GC7 | BHK-R | 7.4 | 42 | 5600 | |
| DFMO | SARS-CoV-2 | Vero-E6 | 230 | 11 | 48 |
To expand on this work, we turned to the MHV model system. As with our experiments with SARS-CoV-2, BHK-21 cells expressing the MHV receptor (BHK-R) (22) were pretreated with various concentrations of DFMO 4 days before infection with an MOI of 0.01. Supernatant was collected 48 h post-infection (hpi), and titers were measured with a plaque assay on BHK-Rs. Viral titers were significantly reduced in a dose-dependent manner, with an IC50 value of 380 μM (Figure 1E). We confirmed that DFMO depleted polyamines in these cells by TLC (Figure 1G), especially levels of putrescine and spermidine, as previously described. (14) To test if DFMO remained antiviral over multiple rounds of infection, BHK-Rs were treated with DFMO and infected at an MOI of 0.01. Supernatant was collected and titered at the indicated time points (Figure 1H). DFMO reduced viral titers compared to the no treatment group up to 72 h, and viral titers in DFMO-treated cells plateaued at a lower level than untreated controls, suggesting that MHV was unable to overcome polyamine depletion over several rounds of replication. We observed that DFMO had modest toxicity in BHK-R cells with a CC50 of 3.8 mM (Figure 1F), giving a selectivity index (SI) of 10. Finally, we measured the relative number of genomes present in the supernatant from the infected cells. Following treatment and infection, we purified, reverse-transcribed, and measured viral genomes by qPCR using specific primers and observed reduced genome levels with DFMO treatment (Figure 1I). Using the data from infectious titers (Figure 1E) and viral genomes (Figure 1I), we calculated the specific infectivity (genomes per PFU) and observed that DFMO treatment increased the ratio of genomes to titer (Figure 1J), similar to previous findings with bunyaviruses and polyamine depletion. (23) Thus, polyamines enhance coronavirus infection and maintain a specific infectivity of MHV.
To address the role of cell type, we tested the ability of DFMO to restrict virus replication in HeLa cells expressing the MHV receptor (HeLa-R). Cells were pretreated with DFMO and infected at an MOI of 0.01 for 48 h. We observed a decline in virus replication, more pronounced than on BHK-R cells, with an IC50 value of approximately 7.0 μM (Figure 1K). We confirmed that DFMO treatment of HeLa-R cells reduced polyamines (Figure 1M) and verified that DFMO was not toxic to these cells (Figure 1L). The CC50 of DFMO in HeLa cells was 16 mM, and thus, the selectivity index was 2300.
Polyamines Facilitate Coronavirus Replication
DFMO restricts virus replication by reducing cellular polyamine pools. (19) However, excess polyamines do not enhance virus replication, according to work based on chikungunya virus. (14) Recent work has suggested that polyamines may serve coronavirus replication. (24) To determine if exogenously supplied polyamines serve an antiviral role, we treated BHK-R cells with increasing doses of the individual polyamines putrescine, spermidine, and spermine, from 1 to 100 μM. We measured cellular viability (Figure 2A) and infected these cells to measure viral titers at 48 hpi (Figure 2B). We observed that the polyamines significantly reduced cellular viability at approximately 5 μM, reflecting prior descriptions of high polyamine concentrations leading to apoptosis. (25,26) Despite this reduction in viability, we observed modestly reduced viral titers (Figure 2B). We measured polyamines in supplemented cells and observed that supplementation with any of the three polyamines resulted in only modest changes in cell-associated polyamines (Figure 2B, lower), as we previously described. (27) The reduced signal with high concentrations of spermidine and spermine likely was an artifact of reduced cellular viability at these doses.
Figure 2
Figure 2. Polyamines facilitate coronavirus replication. (A) BHK-R cells were treated with escalating doses of putrescine, spermidine, and spermine, and cellular viability was measured 24 h later. Calculated CC50 values are in the inset. (B) BHK-R cells treated as in (A) were infected with MHV at an MOI of 0.01 and titered at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (C) BHK-R cells were treated with 1 mM DFMO for 4 days prior to infection with MHV at an MOI of 0.01. At the time of infection, 1 μM polyamines were added to the cells. Viral titers were determined at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (D) MHV was directly incubated with DFMO or 1 μM of the polyamines putrescine (put), spermidine (spd), and spermine (spm) for 24 h and then directly titered. **p < 0.01 by the Student’s t test. Data from at least two independent experiments.
To determine if DFMO-mediated virus restriction was a result of polyamine depletion, we treated BHK-R cells with DFMO and, at the time of infection, supplemented with the individual polyamines putrescine, spermidine, and spermine at 1 μM, a level that did not affect cellular viability (Figure 2A). We observed that DFMO reduced viral titers and that supplementation with any of the individual polyamines rescued virus replication to untreated levels (Figure 2C). We confirmed the presence of these polyamines (Figure 2C, below) and observed that supplementation with any of the individual polyamines results in the accumulation of all three polyamines in the cells. To exclude the possibility that polyamines may directly inactivate viral particles, we directly incubated MHV with DFMO and each of the individual polyamines at 10 μM for 24 h and then titered the incubated virus. We observed that neither DFMO nor polyamines affected infectivity when incubated with virus, as titers were unchanged compared to untreated controls (Figure 2D). In sum, our data suggest that polyamines support virus yield and that supplementation of polyamines supports but does not enhance this yield and can instead be cytotoxic.
Polyamine Depletion Is Prophylactic
DFMO depletes polyamines by inhibiting ODC1, though time is required to deplete cellular polyamine pools and subsequently reduce viral titers. (14) Ideally, the treatment with antivirals commences after the initiation of infection, though identification of soon-to-be-infected individuals is impractical. Thus, to determine if we could treat cells with DFMO at the time of infection, which would be more clinically relevant, we determined whether DFMO suppresses plaque development when present in overlay media, as previously described. (19) We observed no changes in plaque size (Figure 3A) or morphology (Figure 3B) with DFMO supplementation to the media, suggesting that DFMO was not sufficiently antiviral when applied to cells after infection and highlighting that DFMO must be applied prophylactically to reduce coronavirus replication. We observed a small, though statistically insignificant change in HeLa-R cells (Figure 3C).
Figure 3
Figure 3. Polyamine depletion is antiviral prophylactically. Confluent monolayers of BHK-R cells were treated with DFMO at the time of infection with MHV and overlaid with agarose to form plaques. (A) Plaques were quantified 2 days later. (B) Representative plaques are shown from (A). (C) HeLa-R cells were infected and treated as in (A) and the plaque area was measured. (D) BHK-R cells were treated with 1.5 mM DFMO in one, three, or four doses prior to infection with MHV at an MOI of 0.01. Viral titers were determined by plaque assay at 48 hpi. (E) Cells were treated with 1 mM DFMO and then subsequently refed with fresh medium not containing DFMO 24 h before infection with MHV at an MOI of 0.01. Titers were determined at 24 hpi. **p < 0.01 and ***p < 0.001 by the Student’s t test. Representative data from at least three independent experiments. Individual plaque sizes are indicated in (A) and (C).
The use of DFMO as an anti-trypanosomal agent requires repeated dosing to reach a serum concentration high enough to inhibit trypanosome growth. (11,12,28) To mimic this, we treated BHK-R cells with 1.5 mM DFMO in a single treatment, three 500 μM treatments, or four 375 μM treatments equally spaced over the 4 days prior to infection. We observed that cells receiving multiple doses of the drug reduced cellular capacity to support virus production, similar to when the drug was delivered in a single dose (Figure 3D), suggesting that repeated dosing is as effective as a single drug treatment in vitro. Finally, we tested whether the antiviral effects of DFMO were reversed by removing the drug and replenishing cells with fresh media. We treated cells with 1 mM for 4 days and then removed and replaced the media with fresh media without drug for 24 h before infection. When we measured viral titers, we observed the DFMO treatment reduced infectious virus production, and this effect was completely reversed by replenishing the cells with fresh media, suggesting that DFMO’s antiviral effect can be reversed by removing the drug.
Polyamines Facilitate Coronavirus Binding and Entry
We previously demonstrated that enteroviruses, flaviviruses, and bunyaviruses rely on polyamines for viral attachment and entry. (15) To test whether coronaviruses similarly rely on polyamines for attachment and entry, we performed a binding assay in cells treated with DFMO. We treated BHK-R cells with DFMO for 4 days prior to infection with 1000 pfu. After 5 min, unbound virus was washed away with PBS and cells were overlaid with agarose for a plaque assay, lacking DFMO in the overlay. Thus, DFMO is present only during binding of MHV to target cells and not for any other portion of the viral life cycle. Plaques were then allowed to form and were enumerated, representing bound and entered infectious virus. We observed that the attachment and plaque formation was significantly inhibited with DFMO treatment (Figure 4A, representative plaques for NT and 1 mM in Figure 4B). We expanded these results by infecting DFMO-treated BHK-R cells with MHV for 5 min, washing away unbound virus, and quantifying bound virus by qRT-PCR. We observed that, similar to our plaque formation assays, bound viral genomes were significantly reduced with DFMO treatment (Figure 4C). We confirmed that the cells expressed the MHV receptor (CECAM-1) by immunofluorescence (Figure 4D), regardless of DFMO treatment, suggesting that DFMO treatment was not directly reducing receptor availability. We recapitulated these results with HeLa-R cells and observed a similar reduction in virus binding with DFMO treatment (Figure 4E).
Figure 4
Figure 4. Polyamines facilitate coronavirus cellular attachment. Confluent BHK-R cells were treated with DFMO prior to the MHV binding assay. Virus was inoculated on cells for 5 min prior to removal, wash, and agarose overlay in media without DFMO. Plaques formed were quantified (A), and representative images from 1 mM DFMO treatment are shown (B). (C) BHK-R cells were treated as in (A), but after washing unattached virus, attached virus and cells were collected for RNA purification, reverse transcription, and viral genome quantitation by qPCR. Attached genomes were normalized to input virus. (D) MHV receptor (CECAM-1) was visualized on untreated and 1 mM DFMO treated cells. (E) HeLa-R cells were treated with 1 mM DFMO, infected with MHV, and processed as in (C). (F) Vero-E6 cells were treated with DFMO and analyzed for binding ability of HCoV-NL63 as in (C). (G) ACE2 expression on Vero-E6 cells was confirmed by immunofluorescence. (H) Pseudotyped viral particles with no viral glycoproteins or the SARS-CoV-2 spike were tested for their ability to bind and enter Vero-E6 cells. (I) Spike protein was confirmed to be present on pseudotyped virus by western blot. (J) Vero-E6 cells were treated with DFMO and infected with SARS-CoV-2 pseudoparticles as in (C). *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
To expand to human coronaviruses, we tested HCoV-NL63 for binding to DFMO-treated Vero-E6 cells. Again, we observed that the attachment was sensitive to DFMO treatment (Figure 4E). Again, we noted no change in viral receptor expression (ACE2, Figure 4G). Taking advantage of a pseudoparticle system as previously described (29) and adapted to SARS-CoV-2, we measured the attachment of SARS-CoV-2 to DFMO-treated cells. We verified that these pseudoparticles were competent to bind susceptible cells compared to “bald” particles, lacking viral spike (Figure 4H), and we confirmed the presence of the spike protein on purified particles by western blot (Figure 4I). We observed a reduction in cellular attachment of SARS-CoV-2 pseudoparticles in a dose-dependent manner (Figure 4H). Thus, human coronaviruses, including pandemic SARS-CoV-2, are sensitive to polyamine depletion and rely on polyamines for efficient cellular attachment.
Additional Molecules Targeting the Polyamine and Hypusination Pathway Quell Coronavirus Infection
Several pharmaceuticals target polyamine biosynthesis (Figure 5A) and exhibit antiviral activity, (16,19) though coronaviruses have not been thoroughly tested. Additionally, an offshoot of the polyamine pathway, the cellular hypusination pathway, in which spermidine is conjugated to eIF5A, plays roles in cellular translation, (30) which is critical for filovirus replication. (16,31) To determine if additional compounds targeting the polyamine and hypusination pathways exhibited activity against coronaviruses, we treated cells with increasing doses of inhibitors of these pathways. We used the molecules diethylnorspermidine (DENSpm; enhances spermidine–spermine acetyltransferase [SAT1] activity and polyamine breakdown), GC7 (inhibits deoxyhypusine synthase [DHPS] to prevent eIF5A hypusination), and deferiprone and ciclopirox (DEF and CPX; inhibit deoxyhypusine hydroxylase [DOHH]) because they target distinct steps in the polyamine pathway. DENSpm significantly reduced polyamine levels and MHV titers at 10 μM (Figure 5B) with an IC50 of 1.6 μM. We observed minimal toxicity (Figure 5C) with a CC50 value of 35 μM resulting in a selectivity index of 23. Because DENSpm activates SAT1 to deplete polyamines, its action is much more rapid than DFMO. Thus, we tested whether DENSpm exhibited antiviral activity after the initiation of infection. Cells were treated with DENSpm at times before and after infection, and virus titers were measured at 48 hpi. We observed that DENSpm was most effective when treatment was initiated before infection; however, significant antiviral activity was maintained when DENSpm was added as late as 4 hpi (Figure 5D). Finally, we tested the hypusination inhibitors GC7, DEF, and CPX. We observed in all cases that these molecules significantly reduced MHV replication in a dose-dependent manner (Figure 5E–G) and that cytotoxicity was minimal (Figure 5H; summarized in Table 1). Thus, molecules targeting an additional branch of the polyamine biosynthetic pathway exhibit anti-coronavirus activity.
Figure 5
Figure 5. Additional molecules targeting the polyamine pathway quell coronavirus infection. (A) Schematic of the polyamine and hypusination pathway. (B) HeLa-R cells were treated with escalating doses of DENSpm for 16 h prior to infection with MHV at an MOI of 0.01. Viral titers were determined at 48 hpi (upper). Polyamine levels were measured by chromatography (lower). (C) Treated cells were assayed for viability after 16 h of treatment with DENSpm. (D) HeLa-R cells were treated with DENSpm at the indicated times before and after infection with MHV at an MOI of 10. Viral titers were determined at 48 hpi. BHK-R cells were treated with (E) CPX, (F) DEF, and (G) GC7 for 24 h prior to infection with MHV at an MOI of 0.01. Viral titers were determined at 48 hpi. (H) BHK-R cells treated with CPX, DEF, and GC7 were analyzed for cell viability after 24 h of treatment. *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
Discussion
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Polyamines are crucial to virus infection; however, their role in coronavirus replication was previously unknown. Here, we demonstrated that polyamines are essential to coronavirus infection and that attachment is at least one of the steps affected by polyamine depletion. We previously demonstrated that enteroviruses are similarly sensitive to polyamine depletion and rely on these polyamines for attachment. (15) This phenotype is conserved among several viral families, suggesting that polyamines may mediate a common attachment factor. Whether polyamines directly facilitate attachment by mediating virus–cell connections is unclear, and future work will be needed to delineate precisely how polyamines affect attachment. In addition to attachment, we observed that inhibitors of the hypusination pathway impact virus replication, suggesting that polyamines may facilitate infection through eIF5A hypusination, as observed for Ebolavirus. (16) Given the role of hypusinated eIF5A in translation, inhibitors of this pathway may directly inhibit viral translation or may inhibit the translation of a cellular factor essential to virus replication. Regardless, targeting polyamines may affect multiple steps of infection, and additional work will be needed to uncover additional mechanisms.
Polyamines play several important roles in the cell, and recent work has highlighted pathways that may modulate virus replication. Spermidine, as a substrate for eIF5A hypusination, enhances cellular autophagy, (32) and recent work has suggested that spermidine-enhanced autophagy limits SARS-CoV-2 replication. (24) In contrast, our results suggest that spermidine does not inhibit or restrict virus replication at concentrations up to 100 μM. We do observe that spermine supplementation to cells reduces virus replication; however, we also observe significant cytotoxicity with spermine treatment, likely the reason for reduced titers. Additionally, we observed that DFMO treatment reduced polyamine content within cells, even at concentrations that did not affect viral titers. These data suggest that coronaviruses require a low concentration of polyamines for robust replication. In fact, bunyaviruses and alphaviruses replicate with supplementation of 100 nM polyamines in polyamine depleted cells. (14,33) Thus, these viruses may have evolved mechanisms to take advantage of polyamines even at low concentrations. This has implications for the molecular interactions between polyamines and coronaviruses, and perhaps coronaviruses evolved a strong affinity for polyamines. This also has implications for infection in an organism, as polyamines are transported between cells. In cancer therapies, polyamine transporter inhibitors can be used in conjunction with polyamine depletion agents to prevent both polyamine synthesis and internalization. While this has not been tested in the context of virus infection, the combination of inhibitors of synthesis and transport may more effectively reduce virus replication. Precisely how polyamines play into cellular signaling and metabolic pathways to affect virus replication is not clear; however, the complex interplay between metabolism and virus replication affords several opportunities to target virus replication with small molecule inhibitors.
With the current SARS-CoV-2 outbreak and the resulting coronavirus disease of 2019 (COVID-19), novel antivirals are urgently needed. Remdesivir has shown significant potential clinically, (34−37) and additional clinical testing will determine if this molecule can effectively treat infected individuals. Our work with DFMO and polyamine depletion highlights another promising intervention. An important limitation of our study is the relatively high concentration of drug required to reduce coronavirus infection. Given this caveat, the possibility of off-target antiviral effects cannot be ignored. However, we show that DFMO’s antiviral effects are reversed by the addition of exogenous polyamines, suggesting specificity. Our prior results with chikungunya, Zika, Coxsackie, and Rift Valley fever viruses have demonstrated that low micromolar concentrations of inhibitor are sufficient to reduce virus replication. Thus, coronaviruses may be less reliant on polyamines than other viral families, though this requires significant further investigation.
Importantly, we observe that polyamine depletion functions prophylactically to reduce virus replication and may not reduce viral titers after the initiation of infection. Given the limits to our in vitro studies, we cannot say for certain whether polyamine depletion in a human could reduce virus replication or improve clinical outcome when treatment commences after exposure. However, polyamine depletion may be most effective as a prophylactic, preventing infection of vulnerable populations. With the significant spread of SARS-CoV-2, protecting vulnerable populations, including the elderly and immunocompromised, is essential to reducing mortality. Further, prophylactically treating frontline workers, including medical staff, could reduce the spread of the virus in a hospital setting. Currently, DFMO is approved for human use as a topical and intravenous agent. Nebulization and direct lung delivery, if effective at depleting polyamines in the lung, would be a benefit for reducing virus burden, but this remains untested. Whether DFMO or other polyamine-targeting drugs are sufficient to do this is unclear and requires significantly more investigation, including in animal models.
Methods
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Cell Culture
Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) with bovine serum and penicillin–streptomycin at 37 °C and 5% CO2. Vero cells were obtained through BEI Resources, and VeroE6 cells were obtained through ATCC (CRL-1586). Vero and HeLa-R cells were supplemented with 10% new-born calf serum (NBCS; Thermo-Fischer). BHK-R cells were kindly provided by Dr. Susan Baker and were supplemented with 10% fetal bovine serum (FBS; Thermo-Fischer). HEK293T cells were grown in 10% FBS.
Infection and Enumeration of Viral Titers
MHV-A59 was derived from the first passage of virus in BHK-R cells from an infectious clone. SARS-CoV-2, isolate BetaCoV/France/IDF0372/2020 C2, was supplied by the National Reference Centre for Respiratory Viruses hosted at Institut Pasteur (Paris, France). The human sample from which this strain was isolated was provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France. Viral stocks were prepared by propagation in Vero E6 cells in DMEM supplemented with 2% FBS. All experiments involving live SARS-CoV-2 were performed in compliance with the Institut Pasteur guidelines for Biosafety Level 3 work. For all infections, DFMO was maintained throughout the infection as designated. Viral stocks were maintained at −80 °C. For infection, virus was diluted in serum-free DMEM for a multiplicity of infection (MOI) of 0.01 on BHK-R cells, unless otherwise indicated. Viral inoculum was added to the cells, and supernatants were collected at specified time points. To quantify viral titers via plaque assay, dilutions of cell supernatant were prepared in serum-free DMEM and used to inoculate confluent monolayers of BHK-R cells for 10 to 15 min at 37 °C. Cells were overlain with 0.8% agarose in DMEM containing 2% FBS. MHV-A59 samples were incubated for 2 days at 37 °C. Cells were fixed with 4% formalin and revealed with crystal violet solution (10% crystal violet; Sigma-Aldrich). Plaques were enumerated and used to back-calculate the number of plaque forming units (PFU) per milliliter of collected volume.
Drug Treatments
Difluoromethylornithine (DFMO; TargetMol) was diluted to a 100 mM solution in sterile PBS. For DFMO treatments, cells were trypsinized (Zymo Research) and reseeded with fresh medium supplemented with 2% FBS. Cells were treated with 1 mM DFMO unless otherwise indicated. Cells were incubated with DFMO for 96 h to allow for the depletion of polyamines. Experiments involving polyamine rescues were performed using 1 μM polyamines (Sigma-Aldrich) added to either the cell supernatant or viral inoculum as indicated.
Thin Layer Chromatography of Polyamines
Polyamines were separated by thin layer chromatography as previously described. (38) For all samples, cells were treated as described prior to being trypsinized and centrifuged. Pellets were washed with PBS and then resuspended in 200 μL of 2% perchloric acid. Samples were then incubated overnight at 4 °C. Supernatant or 1 μmole of putrescine, spermidine, or spermine standards was combined with 5 mg/mL dansyl chloride (Sigma-Aldrich) in acetone and saturated sodium bicarbonate. Samples were incubated in the dark overnight at room temperature. Excess dansyl chloride was cleared by incubating the reaction with proline (Sigma-Aldrich). Dansylated polyamines were extracted with toluene (Sigma-Aldrich) and centrifuged. The sample was added in spots to the silica gel matrix TLC plates (Sigma-Aldrich) and exposed to ascending chromatography with 1:1 cyclohexane/ethyl acetate. The plate was dried and visualized via exposure to UV.
Plaque Formation Attachment Assay
BHK-R cells were seeded in 6-well plates and grown to confluence in DMEM with 2% FBS. The cells were treated for 96 h with varying concentrations of DFMO. After the 96 h DFMO treatment, the media were aspirated from the cells and replaced with 500 μL of serum free media containing 1000 PFU MHV. The infected cells were incubated at room temperature or on ice for 5 min. Cells were washed 3× with PBS and then overlaid with 0.8% agarose containing DMEM with 2% FBS. The plates were incubated at 37 °C for 2 days for plaques to develop. The cells were fixed with 4% formalin, and the plaques were visualized with crystal violet staining.
Plaque Size Measurement
BHK-R or HeLa-R cells were seeded in 6-well plates and grown to confluence. Approximately 100 PFU of MHV-A59 was diluted in a 500 μL inoculum of serum free DMEM. The inoculum was incubated on the cells for approximately 30 min at 37 °C. After 30 min, an overlay of 8 mL of 0.8% agarose containing DMEM with 2% FBS (BHK-R) or NBCS (HeLa-R) was added to each well. The dishes were incubated at 37 °C for 2 days to allow plaque formation. The cells were fixed with 4% formalin, and the agarose plugs were removed. The fixed cells were stained with crystal violet. Plaque size was determined using ImageJ software. (39)
Immunofluorescence Imaging
Cells grown on coverslips were either treated with 1 mM DFMO or untreated. Cells were fixed with 4% formalin for 15 min, washed with PBS, permeabilized, and blocked with 0.2% Triton X-100 and 2% BSA in PBS (blocking solution) for 30 min at room temperature (RT). Cells were sequentially incubated as follows: primary rabbit anti-hACE2 (Invitrogen, SN0754) (Vero E6) or rabbit anti-mCEACAM (BHK-R) antibodies (1:500) with blocking for 2 h at room temperature. Cells were subsequently washed then incubated with secondary goat anti-rabbit antibodies (1:500 with blocking, 30 min, RT). Mounting media with DAPI was used to visualize nuclei. Samples were imaged with a Zeiss Axio Observer 7 with Lumencor Spectra X LED light system and a Hamamatsu Flash 4 camera using appropriate filters using Zen Blue software with a 40× objective.
Western Blot
Samples were collected with Bolt LDS Buffer and Bolt Reducing Agent (Invitrogen) and run on polyacrylamide gels. Gels were transferred using the iBlot 2 Gel Transfer Device (Invitrogen). Membranes were probed with primary antibodies for mouse anti-rhodopsin (EMD Millipore, MAB5356), anti-VSV-M (23H12), and anti-mouse IgG (Goat, HRP-Labeled, NEF822001EA). Membranes were treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and visualized on a ProteinSimple FluorChem E imager.
RNA Purification and cDNA Synthesis
Media were cleared from cells, and Trizol reagent (Zymo Research) was added directly. Lysate was then collected, and RNA was purified through a Zymo RNA extraction kit. Purified RNA was subsequently used for cDNA synthesis using High Capacity cDNA Reverse Transcription Kits (Thermo-Fischer), according to the manufacturer’s protocol, with 10–100 ng of RNA and random hexamer primers.
qPCR-Based Attachment Assay
BHK-R, HeLa-R, and Vero cells were seeded at 4 × 104 cells per well in 24-well plates in DMEM with either 2% FBS or NBCS. The cells were treated for 96 h with varying concentrations of DFMO. After 96 h, the media were aspirated from the cells and replaced with 100 μL of serum free media containing virus. The infected cells were incubated for 5 min at room temperature or on ice. The cells were then washed 1× with PBS, and then, 200 μL of Trizol was added to the cells. The RNA was extracted with the Zymo RNA extraction kit, converted to cDNA, and quantified by real-time PCR with SYBR Green (DotScientific) using the one-step protocol QuantStudio 3 (ThermoFisher Scientific). Relative genomes were calculated using the ΔCT method, normalized to the β-actin qRT-PCR control, and calculated as the fraction of the unwashed samples. Primer sequences are as follows: NL63, 5′-TGT-CAA-CGA-GGT-TTT-GCA-TTA-AAT-3′ (F) and 5′-ACT-GGC-CTA-CCA-TTG-TGT-GTA-AGA-3′ (R); MHV, 5′-ATC-CTC-AAG-AAG-ACC-ACT-TGG-GCT-GAC-3′ (F) and 5′-GAG-TAA-TGG-GGA-ACC-ACA-CTC–CCG-3′ (R); β-actin, 5′-CAC-TCT-TCC-AGC-CTT-CCT-TC-3′ (F) and 5′-GTA-CAG-GTC-TTT-GCG-GAT-GT-3′ (R). Primers were verified for linearity using 8-fold serial diluted cDNA and checked for specificity via melt curve analysis. SARS-CoV-2 genomes were detected using a Taqman assay (IP4 set) as described on the WHO Web site (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2)
Synthesis of SARS-CoV-2 Pseudovirus
293T cells were seeded into a 150 mm tissue culture dish 24 h prior to transfection. Cells were transfected with a SARS-CoV-2 spike using LipoD293 and following the manufacture’s protocol. After 6 h, the media were removed and replaced with fresh, prewarmed 10% FBS media. The following day, cells were infected with a 1 to 100 dilution of Fluc-VSV-Junin virus and allowed to infect for 2 h. After the 2 h, the cells were washed 2× with serum free DMEM, and fresh, prewarmed media were placed onto the cells. On the following day, cell supernatant was collected and spun down via differential centrifugation (300g for 10 min at 4 °C, 4500g for 10 min at 4 °C) and stored at −80 °C.
Pseudovirus Attachment Assay
Vero E6 cells were seeded at 4 × 104 in 24-well plates in DMEM with 2% NBCS. Cells were then treated with varying concentrations of DFMO for 96 h. After 96 h, the media were aspirated from the cells and replaced with 100 μL of serum free media containing pseudovirus. The infected cells were incubated for 5 min at room temperature. Pseudovirus was then aspirated off and washed 1× with PBS (bound), and new, prewarmed 2% NBCS media were added to the cells and allowed to sit overnight (input and bound). The following day, the media were aspirated from the cells, and the cells were then washed with 1× PBS. Subsequently, cells were then lysed with 75 μL of 1× Pierce Lysis Buffer (Promega) for 10 min. The lysed samples were then transferred to a white, opaque 96-well plate. 150 μL of luciferase was added to wells and read with a GlowMax luminometer. The percent bound was calculated by normalizing luciferase values to the input.
Genome/PFU Ratio
Cells were infected with the MHV-A59 at an MOI of 0.01. Supernatant was collected 48 hpi. The infectious virus (PFU) was quantified via plaque assay as described. RNA was extracted, reverse-transcribed, and quantified with qPCR as described above.
Statistical Analysis
Prism 6 (GraphPad) was used to generate graphs and perform statistical analysis. For all analyses, the two-tailed Student’s t test was used to compare groups, unless otherwise noted, with a = 0.05. For tests of sample proportions, p values were derived from the calculated Z scores with two tails and a = 0.05.
Author Information
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- Bryan C. Mounce - Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United States;
http://orcid.org/0000-0003-1710-5362;
- Tom Gallagher - Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, United States;http://orcid.org/0000-0002-8601-5961
Acknowledgments
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We thank Susan Baker for generous assistance with the MHV system and for providing the virus and cells as well as Ivana Kuo for microscopy assistance. We also thank Susan Uprichard for critical discussion of the data. E.S.-L. acknowledges funding from the INCEPTION program (Investissements d’Avenir grant ANR-16-CONV-0005), from the Institut Pasteur corona task force, and from the Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (grant no. ANR-10-LABX-62-IBEID).
References
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This article references 39 other publications.
- 1de Wit, E., van Doremalen, N., Falzarano, D., and Munster, V. J. (2016) SARS and MERS: Recent Insights into Emerging Coronaviruses. Nat. Rev. Microbiol. 14 (8), 523– 534, DOI: 10.1038/nrmicro.2016.81Google Scholar1SARS and MERS: recent insights into emerging coronavirusesde Wit, Emmie; van Doremalen, Neeltje; Falzarano, Darryl; Munster, Vincent J.Nature Reviews Microbiology (2016), 14 (8), 523-534CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The continuing introductions of MERS-CoV from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. Scientific advancements since the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) pandemic allowed for rapid progress in our understanding of the epidemiol. and pathogenesis of MERS-CoV and the development of therapeutics. In this Review, we detail our present understanding of the transmission and pathogenesis of SARS-CoV and MERS-CoV, and discuss the current state of development of measures to combat emerging coronaviruses.
- 2Gralinski, L. E. and Menachery, V. D. (2020) Return of the Coronavirus: 2019-NCoV. Viruses 12 (2), 135, DOI: 10.3390/v12020135Google ScholarThere is no corresponding record for this reference.
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- 4Pyrc, K., Dijkman, R., Deng, L., Jebbink, M. F., Ross, H. A., Berkhout, B., and van der Hoek, L. (2006) Mosaic Structure of Human Coronavirus NL63, One Thousand Years of Evolution. J. Mol. Biol. 364 (5), 964– 973, DOI: 10.1016/j.jmb.2006.09.074Google Scholar4Mosaic Structure of Human Coronavirus NL63, One Thousand Years of EvolutionPyrc, Krzysztof; Dijkman, Ronald; Deng, Lea; Jebbink, Maarten F.; Ross, Howard A.; Berkhout, Ben; van der Hoek, LiaJournal of Molecular Biology (2006), 364 (5), 964-973CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is assocd. with croup in children. We report here the complete genome sequence of two HCoV-NL63 clin. isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, resp., and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic anal. revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Addnl., employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be ∼ 3 × 10-4 substitutions per site per yr. Using this evolutionary rate we detd. that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.
- 5van der Hoek, L., Ihorst, G., Sure, K., Vabret, A., Dijkman, R., de Vries, M., Forster, J., Berkhout, B., and Uberla, K. (2010) Burden of Disease Due to Human Coronavirus NL63 Infections and Periodicity of Infection. J. Clin. Virol. 48 (2), 104– 108, DOI: 10.1016/j.jcv.2010.02.023Google Scholar5Burden of disease due to human coronavirus NL63 infections and periodicity of infectionvan der Hoek Lia; Ihorst Gabriele; Sure Klaus; Vabret Astrid; Dijkman Ronald; de Vries Michel; Forster Johannes; Berkhout Ben; Uberla KlausJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology (2010), 48 (2), 104-8 ISSN:.BACKGROUND: The disease burden caused by recently identified respiratory viruses like HCoV-NL63 is unknown. OBJECTIVES: We determined the burden of disease due to HCoV-NL63 infections using the population-based PRI.DE cohort of children under the age of 3 with lower respiratory tract infections (LRTIs). STUDY DESIGN: In total 1756 respiratory samples, from hospitalized children or children who visited the outpatient clinic, were tested for HCoV-NL63. Sampling covered a period of 2 years and the frequency of infection in different years was compared to other Western European studies that tested for this virus in 2 or more consecutive years. RESULTS: Sixty-nine samples were HCoV-NL63 positive, 35 were with high loads, and of these 25 were single HCoV-NL63 infections. Based on the number of children with high HCoV-NL63 infection and no additional infection, the overall annual incidence in outpatients was 7 per 1000 children per year (95% confidence interval (CI) 3-13 per 1000 children per year), which can be extrapolated to an absolute number of 16,929 visits to the physician due to an HCoV-NL63 infection in Germany per year. The estimated hospitalization rate is 22 per 100,000 children (95% CI: 7-49 per 100,000 children per year). This number reflects 522 HCoV-NL63 children in Germany per year. A large year-to-year difference in HCoV-NL63 infection frequency was observed. Combining these data with those of other studies in Western Europe revealed that HCoV-NL63 infections follow a 2-year inter-epidemic period with peaks of infection in the winters of 2000/2001, 2002/2003 and 2004/2005 (p<0.0001). CONCLUSIONS: HCoV-NL63 infection in children below 3 years of age often requires a visit to the physician in an outpatient clinic, especially during peak-years, but hospitalizations are relatively infrequent.
- 6Brown, A. J., Won, J. J., Graham, R. L., Dinnon, K. H., Sims, A. C., Feng, J. Y., Cihlar, T., Denison, M. R., Baric, R. S., and Sheahan, T. P. (2019) Broad Spectrum Antiviral Remdesivir Inhibits Human Endemic and Zoonotic Deltacoronaviruses with a Highly Divergent RNA Dependent RNA Polymerase. Antiviral Res. 169, 104541, DOI: 10.1016/j.antiviral.2019.104541Google Scholar6Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymeraseBrown, Ariane J.; Won, John J.; Graham, Rachel L.; Dinnon, Kenneth H., III; Sims, Amy C.; Feng, Joy Y.; Cihlar, Tomas; Denison, Mark R.; Baric, Ralph S.; Sheahan, Timothy P.Antiviral Research (2019), 169 (), 104541CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV.
- 7Sheahan, T. P., Sims, A. C., Graham, R. L., Menachery, V. D., Gralinski, L. E., Case, J. B., Leist, S. R., Pyrc, K., Feng, J. Y., Trantcheva, I., Bannister, R., Park, Y., Babusis, D., Clarke, M. O., Mackman, R. L., Spahn, J. E., Palmiotti, C. A., Siegel, D., Ray, A. S., Cihlar, T., Jordan, R., Denison, M. R., and Baric, R. S. (2017) Broad-Spectrum Antiviral GS-5734 Inhibits Both Epidemic and Zoonotic Coronaviruses. Sci. Transl. Med. 9 (396), eaal3653, DOI: 10.1126/scitranslmed.aal3653Google ScholarThere is no corresponding record for this reference.
- 8Gerner, E. W. and Meyskens, F. L. (2004) Polyamines and Cancer: Old Molecules, New Understanding. Nat. Rev. Cancer 4 (10), 781– 792, DOI: 10.1038/nrc1454Google Scholar8Polyamines and cancer: Old molecules, new understandingGerner, Eugene W.; Meyskens, Frank L., Jr.Nature Reviews Cancer (2004), 4 (10), 781-792CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. The amino-acid-derived polyamines have long been assocd. with cell growth and cancer, and specific oncogenes and tumor-suppressor genes regulate polyamine metab. Inhibition of polyamines synthesis has proven to be generally ineffective as an anticancer strategy in clin. trials, but it is a potent cancer chemoprevention strategy in preclin. studies. Clin. trials, with well-defined goals, are now underway to evaluate the chemopreventive efficacy of inhibitors of polyamine synthesis in a range of tissues.
- 9Frugier, M., Florentz, C., Hosseini, M. W., Lehn, J. M., and Giegé, R. (1994) Synthetic Polyamines Stimulate in Vitro Transcription by T7 RNA Polymerase. Nucleic Acids Res. 22 (14), 2784– 2790, DOI: 10.1093/nar/22.14.2784Google Scholar9Synthetic polyamines stimulate in vitro transcription by T7 RNA polymeraseFrugier, Magali; Florentz, Catherine; Hosseini, Mir Wais; Lehn, Jean Marie; Giege, RichardNucleic Acids Research (1994), 22 (14), 2784-90CODEN: NARHAD; ISSN:0305-1048.The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compds. used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and no. of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulated transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concn. For each compd., an optimal concn. could be defined; above this concn., transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compd. called [38]N6C10.
- 10Mandal, S., Mandal, A., Johansson, H. E., Orjalo, A. V., and Park, M. H. (2013) Depletion of Cellular Polyamines, Spermidine and Spermine, Causes a Total Arrest in Translation and Growth in Mammalian Cells. Proc. Natl. Acad. Sci. U. S. A. 110 (6), 2169– 2174, DOI: 10.1073/pnas.1219002110Google Scholar10Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cellsMandal, Swati; Mandal, Ajeet; Johansson, Hans E.; Orjalo, Arturo V.; Park, Myung HeeProceedings of the National Academy of Sciences of the United States of America (2013), 110 (6), 2169-2174, S2169/1-S2169/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromol. synthesis in vitro, their specific functions in vivo are poorly understood. Here, the authors investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N1-acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with green fluorescent protein (GFP) and SAT1 vectors suppressed GFP expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect was most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggested a direct role for polyamines in translation initiation. The data provided strong evidence for a primary function of the polyamines, spermidine and spermine, in translation in mammalian cells.
- 11Burri, C. and Brun, R. (2003) Eflornithine for the Treatment of Human African Trypanosomiasis. Parasitol. Res. 90 (S1), S49– 52, DOI: 10.1007/s00436-002-0766-5Google ScholarThere is no corresponding record for this reference.
- 12Milord, F., Pépin, J., Loko, L., Ethier, L., and Mpia, B. (1992) Efficacy and Toxicity of Eflornithine for Treatment of Trypanosoma Brucei Gambiense Sleeping Sickness. Lancet 340 (8820), 652– 655, DOI: 10.1016/0140-6736(92)92180-NGoogle Scholar12Efficacy and toxicity of eflornithine for treatment of Trypanosoma brucei gambiense sleeping sicknessMilord F; Pepin J; Loko L; Ethier L; Mpia BLancet (London, England) (1992), 340 (8820), 652-5 ISSN:0140-6736.The usual first-line treatment for Trypanosoma brucei gambiense sleeping sickness is melarsoprol, but when that fails the outlook has hitherto been grim. The polyamine synthesis inhibitor eflornithine (difluoromethylornithine, DFMO) has emerged as an alternative therapy. 207 patients with late-stage T b gambiense sleeping sickness were treated in rural Zaire with three different regimens of DFMO in an open-trial design. During treatment, trypanosomes disappeared from the CSF of all 87 patients in whom parasites had been seen before DFMO administration, and there was a sharp fall in CSF white cell count from a mean of 186/microliters to 21/microliters. 152 patients have been followed for at least a year after DFMO treatment, and only 13 (9%) have relapsed. Treatment failures were more common in children less than 12 years, among patients treated with oral DFMO only, and among patients who received DFMO as the initial treatment of their recently diagnosed trypanosomiasis. Toxicity was acceptable. Only 4 patients died during or shortly after treatment. Bone marrow suppression resulting in anaemia (43%) or leucopenia (53%) was common but bore little consequence. This open trial shows that DFMO is as active as and possibly less toxic than melarsoprol. For economic and logistic reasons DFMO may not be the first-choice therapy in rural Africa but for the vast majority of patients who relapse after melarsoprol DFMO will be curative.
- 13Shapiro, J. and Lui, H. (2001) Vaniqa - Eflornithine 13.9% Cream. Skin Therapy Letter 6 (7), 1Google Scholar13Vaniqa--eflornithine 13.9% creamShapiro J; Lui HSkin therapy letter (2001), 6 (7), 1-3, 5 ISSN:1201-5989.Eflornithine HCl 13.9% cream is the first topical prescription treatment to be approved by the US FDA for the reduction of unwanted facial hair in women. It irreversibly inhibits ornithine decarboxylase (ODC), an enzyme that catalyzes the rate-limiting step for follicular polyamine synthesis, which is necessary for hair growth. In clinical trials eflornithine cream slowed the growth of unwanted facial hair in up to 60% of women. Improvement occurs gradually over a period of 4-8 weeks or longer. Most reported adverse reactions consisted of minor skin irritation.
- 14Mounce, B. C., Poirier, E. Z., Passoni, G., Simon-Loriere, E., Cesaro, T., Prot, M., Stapleford, K. A., Moratorio, G., Sakuntabhai, A., Levraud, J.-P., and Vignuzzi, M. (2016) Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses. Cell Host Microbe 20 (2), 167– 177, DOI: 10.1016/j.chom.2016.06.011Google Scholar14Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya VirusesMounce, Bryan C.; Poirier, Enzo Z.; Passoni, Gabriella; Simon-Loriere, Etienne; Cesaro, Teresa; Prot, Matthieu; Stapleford, Kenneth A.; Moratorio, Gonzalo; Sakuntabhai, Anavaj; Levraud, Jean-Pierre; Vignuzzi, MarcoCell Host & Microbe (2016), 20 (2), 167-177CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Polyamines are small, pos. charged mols. derived from ornithine and synthesized through an intricately regulated enzymic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these mols. in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies.
- 15Kicmal, T. M., Tate, P. M., Dial, C. N., Esin, J. J., and Mounce, B. C. (2019) Polyamine Depletion Abrogates Enterovirus Cellular Attachment. J. Virol. 93, e01054-19, DOI: 10.1128/JVI.01054-19Google ScholarThere is no corresponding record for this reference.
- 16Olsen, M. E., Filone, C. M., Rozelle, D., Mire, C. E., Agans, K. N., Hensley, L., and Connor, J. H. (2016) Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication. mBio 7 (4), e00882-16, DOI: 10.1128/mBio.00882-16Google ScholarThere is no corresponding record for this reference.
- 17Mounce, B. C., Olsen, M. E., Vignuzzi, M., and Connor, J. H. (2017) Polyamines and Their Role in Virus Infection. Microbiol. Mol. Biol. Rev. 81 (4), e00029-17, DOI: 10.1128/MMBR.00029-17Google ScholarThere is no corresponding record for this reference.
- 18Firpo, M. R. and Mounce, B. C. (2020) Diverse Functions of Polyamines in Virus Infection. Biomolecules 10 (4), 628, DOI: 10.3390/biom10040628Google Scholar18Diverse functions of polyamines in virus infectionFirpo, Mason R.; Mounce, Bryan C.Biomolecules (2020), 10 (4), 628CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)A review. As obligate intracellular parasites, viruses rely on host cells for the building blocks of progeny viruses. Metabolites such as amino acids, nucleotides and lipids are central to viral proteins, genomes, and envelopes, and the availability of these mols. can restrict or promote infection. Polyamines, comprised of putrescine, spermidine, and spermine in mammalian cells, are also crit. for virus infection. Polyamines are small, pos. charged mols. that function in transcription, translation, and cell cycling. Initial work on the function of polyamines in bacteriophage infection illuminated these mols. as crit. to virus infection. In the decades since early virus-polyamine descriptions, work on diverse viruses continues to highlight a role for polyamines in viral processes, including genome packaging and viral enzymic activity. On the host side, polyamines function in the response to virus infection. Thus, viruses and hosts compete for polyamines, which are a crit. resource for both. Pharmacol. targeting polyamines, tipping the balance to favor the host and restrict virus replication, holds significant promise as a broad-spectrum antiviral strategy.
- 19Mounce, B. C., Cesaro, T., Moratorio, G., Hooikaas, P. J., Yakovleva, A., Werneke, S. W., Smith, E. C., Poirier, E. Z., Simon-Loriere, E., Prot, M., Tamietti, C., Vitry, S., Volle, R., Khou, C., Frenkiel, M.-P., Sakuntabhai, A., Delpeyroux, F., Pardigon, N., Flamand, M., Barba-Spaeth, G., Lafon, M., Denison, M. R., Albert, M. L., and Vignuzzi, M. (2016) Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. J. Virol. 90 (21), 9683– 9692, DOI: 10.1128/JVI.01347-16Google Scholar19Inhibition of polyamine biosynthesis is a broad-spectrum strategy against RNA virusesMounce, Bryan C.; Cesaro, Teresa; Moratorio, Gonzalo; Hooikaas, Peter Jan; Yakovleva, Anna; Werneke, Scott W.; Smith, Everett Clinton; Poirier, Enzo Z.; Simon-Loriere, Etienne; Prot, Matthieu; Tamietti, Carole; Vitry, Sandrine; Volle, Romain; Khou, Cecile; Frenkiel, Marie-Pascale; Sakuntabhai, Anavaj; Delpeyroux, Francis; Pardigon, Nathalie; Flamand, Marie; Barba-Spaeth, Giovanna; Lafon, Monique; Denison, Mark R.; Albert, Matthew L.; Vignuzzi, MarcoJournal of Virology (2016), 90 (21), 9683-9692CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compds. show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant pos. charged mols. found in the cell. Here, we describe the antiviral effects of two mols. that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a neg. effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo. These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available mols., such as DFMO.
- 20Compton, S. R., Barthold, S. W., and Smith, A. L. (1993) The Cellular and Molecular Pathogenesis of Coronaviruses. Lab. Anim. Sci. 43 (1), 15– 28Google Scholar20The cellular and molecular pathogenesis of coronavirusesCompton S R; Barthold S W; Smith A LLaboratory animal science (1993), 43 (1), 15-28 ISSN:0023-6764.Coronaviruses cause a wide spectrum of diseases in humans and animals but generally fall into two classes, with respiratory or enteric tropisms. Mouse hepatitis virus (MHV) and rat coronaviruses are the viruses most frequently encountered in the laboratory animal setting. This review focuses primarily on the cellular and molecular aspects of MHV pathogenesis. The high mutation and recombination rates of coronaviruses lead to a diverse, ever-changing population of MHV strains. The spike (S) protein is the most variable coronavirus protein and is responsible for binding to cell surface receptors, inducing cell fusion and humoral and cellular immunity. Differences within the S protein of different MHV strains have been linked to their variable tropisms. Since immunity to MHV is strain-specific, seropositive mice can be reinfected with different strains of MHV. Natural infections with MHV are acute, with persistence occurring at the population level, not within an individual mouse, unless it is immunocompromised. Age, genotype, immunologic status of the mouse, and MHV strain influence the type and severity of disease caused by MHV. Interference with research by MHV has been reported primarily in the fields of immunology and tumor biology and may be a reflection of MHV's capacity to grow in several types of immune cells. While many methods are available to diagnose coronavirus infection, serologic tests, primarily ELISA and IFA, are the most commonly used. MHV is best managed on a preventive basis. Elimination of MHV from a population requires cessation of breeding and halting the introduction of naive mice into the population.
- 21Weiss, S. R., and Leibowitz, J. L. Coronavirus Pathogenesis. In Advances in Virus Research; Maramorosch, K., Shatkin, A. J., and Murphy, F. A., Eds.; Academic Press, 2011; Vol. 81, Chapter 4, pp 85– 164; DOI: 10.1016/B978-0-12-385885-6.00009-2 .Google ScholarThere is no corresponding record for this reference.
- 22Leibowitz, J., Kaufman, G., and Liu, P. (2011) Coronaviruses: Propagation, Quantification, Storage, and Construction of Recombinant Mouse Hepatitis Virus. Curr. Protoc. Microbiol. 21, 15E.1.1– 15E.1.46, DOI: 10.1002/9780471729259.mc15e01s21Google ScholarThere is no corresponding record for this reference.
- 23Mastrodomenico, V., Esin, J. J., Graham, M. L., Tate, P. M., Hawkins, G. M., Sandler, Z. J., Rademacher, D. J., Kicmal, T. M., Dial, C. N., and Mounce, B. C. (2019) Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions. J. Virol. 93, e00530-19, DOI: 10.1128/JVI.00530-19Google ScholarThere is no corresponding record for this reference.
- 24Gassen, N. C., Papies, J., Bajaj, T., Dethloff, F., Emanuel, J., Weckmann, K., Heinz, D. E., Heinemann, N., Lennarz, M., Richter, A., Niemeyer, D., Corman, V. M., Giavalisco, P., Drosten, C., and Müller, M. A. (2020) Analysis of SARS-CoV-2-Controlled Autophagy Reveals Spermidine, MK-2206, and Niclosamide as Putative Antiviral Therapeutics. bioRxiv, 2020.04.15.997254, DOI: 10.1101/2020.04.15.997254 .Google ScholarThere is no corresponding record for this reference.
- 25Igarashi, K. and Kashiwagi, K. (2000) Polyamines: Mysterious Modulators of Cellular Functions. Biochem. Biophys. Res. Commun. 271 (3), 559– 564, DOI: 10.1006/bbrc.2000.2601Google Scholar25Polyamines: Mysterious Modulators of Cellular FunctionsIgarashi, Kazuei; Kashiwagi, KeikoBiochemical and Biophysical Research Communications (2000), 271 (3), 559-564CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)A review, with ∼67 refs. In recent years the functions of polyamines (putrescine, spermidine, and spermine) have been studied at the mol. level. Polyamines can modulate the functions of RNA, DNA, nucleotide triphosphates, proteins, and other acidic substances. A major part of the cellular functions of polyamines can be explained through a structural change of RNA which occurs at physiol. concns. of Mg2+ and K+ because most polyamines exist in a polyamine-RNA complex within cells. Polyamines were found to modulate protein synthesis at several different levels including stimulation of special kinds of protein synthesis, stimulation of the assembly of 30 S ribosomal subunits and stimulation of Ile-tRNA formation. Effects of polyamines on ion channels have also been reported and are gradually being clarified at the mol. level. (c) 2000 Academic Press.
- 26Igarashi, K. and Kashiwagi, K. (2019) The Functional Role of Polyamines in Eukaryotic Cells. Int. J. Biochem. Cell Biol. 107, 104– 115, DOI: 10.1016/j.biocel.2018.12.012
- 27Mastrodomenico, V., Esin, J. J., Qazi, S., Omoba, O. S., Fung, B. L., Khomutov, M. A., Ivanov, A. V., Mukhopadhyay, S., and Mounce, B. C. (2020) Virion-Associated Spermidine Transmits with Rift Valley Fever Virus Particles to Maintain Infectivity. bioRxiv, 2020.01.23.915900, DOI: 10.1101/2020.01.23.915900 .Google ScholarThere is no corresponding record for this reference.
- 28Wolf, J. E., Shander, D., Huber, F., Jackson, J., Lin, C.-S., Mathes, B. M., and Schrode, K. (2007) Eflornithine HCl Study Group. Randomized, Double-Blind Clinical Evaluation of the Efficacy and Safety of Topical Eflornithine HCl 13.9% Cream in the Treatment of Women with Facial Hair. Int. J. Dermatol. 46 (1), 94– 98, DOI: 10.1111/j.1365-4632.2006.03079.xGoogle Scholar28Randomized, double-blind clinical evaluation of the efficacy and safety of topical eflornithine HCl 13.9% cream in the treatment of women with facial hairWolf, John E., Jr.; Shander, Douglas; Huber, Ferdinand; Jackson, Joseph; Lin, Chen-Sheng; Mathes, Barbara M.; Schrode, Kathy; Bergfeld, Wilma F.; Camacho, Francisco; Dobs, Adrian S.; Dunlap, Frank; Hordinsky, Maria; Katz, Irving H.; Lebwohl, Mark; McMichael, Amy; Olsen, Elise A.; Pariser, David M.; Piacquadio, Daniel; Price, Vera; Redmond, Geoffrey P.; Rodriguez, David; Sawaya, Marty E.; Weiss, Jonathan S.; Whiting, David A.; Wilson, David C.; Wolf, John E., Jr.International Journal of Dermatology (2007), 46 (1), 94-98CODEN: IJDEBB; ISSN:0011-9059. (Blackwell Publishing Ltd.)Geater than 40% of women in the USA have unwanted facial hair, a condition that can have a profound impact on a woman's self-esteem, confidence, and overall quality-of-life. Eflornithine is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting enzyme in the synthesis of polyamines, which appears to be crit. to the proliferation of matrix cells in the hair follicle and hence to hair growth. This paper reports the results of two double-blind studies evaluating the efficacy and safety of eflornithine HCl 13.9% cream in women with unwanted facial hair. Eflornithine cream, applied twice daily for 24 wk was well tolerated and significantly reduced the growth of unwanted facial hair as measured by a physician's assessment and hair length/mass. Efficacy was not dependent on the method of hair removal. The treatment was generally well tolerated with some patients experiencing mild, transient burning, stinging, or tingling.
- 29Qing, E., Hantak, M., Perlman, S., and Gallagher, T. (2020) Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection. mBio 11 (1), e02764-19 DOI: 10.1128/mBio.02764-19Google ScholarThere is no corresponding record for this reference.
- 30Park, M. H. and Wolff, E. C. (2018) Hypusine, a Polyamine-Derived Amino Acid Critical for Eukaryotic Translation. J. Biol. Chem. 293 (48), 18710– 18718, DOI: 10.1074/jbc.TM118.003341Google Scholar30Hypusine, a polyamine-derived amino acid critical for eukaryotic translationPark, Myung Hee; Wolff, Edith C.Journal of Biological Chemistry (2018), 293 (48), 18710-18718CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The natural amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) is derived from the polyamine spermidine, and occurs only in a single family of cellular proteins, eukaryotic translation factor 5A (eIF5A) isoforms. Hypusine is formed by conjugation of the aminobutyl moiety of spermidine to a specific lysine residue of this protein. The post-translational synthesis of hypusine involves two enzymic steps, catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for eIF5A activity. Inactivation of either the eIF5A or the DHPS gene is lethal in yeast and mouse, underscoring the vital role of eIF5A hypusination in eukaryotic cell growth and animal development. The long and basic side chain of the hypusine residue promotes eIF5A-mediated translation elongation by facilitating peptide bond formation at polyproline stretches and at many other ribosome-pausing sites. It also enhances translation termination by stimulating peptide release. By promoting translation, the hypusine modification of eIF5A provides a key link between polyamines and cell growth regulation. eIF5A has been implicated in several human pathol. conditions. Recent genetic data suggest that eIF5A haploinsufficiency or impaired deoxyhypusine synthase activity is assocd. with neurodevelopmental disorders in humans.
- 31Olsen, M. E., Cressey, T. N., Mühlberger, E., and Connor, J. H. (2018) Differential Mechanisms for the Involvement of Polyamines and Hypusinated EIF5A in Ebola Virus Gene Expression. J. Virol. 92, e01260-18, DOI: 10.1128/JVI.01260-18Google ScholarThere is no corresponding record for this reference.
- 32Zhang, H., Alsaleh, G., Feltham, J., Sun, Y., Napolitano, G., Riffelmacher, T., Charles, P., Frau, L., Hublitz, P., Yu, Z., Mohammed, S., Ballabio, A., Balabanov, S., Mellor, J., and Simon, A. K. (2019) Polyamines Control EIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence. Mol. Cell 76 (1), 110– 125.E9, DOI: 10.1016/j.molcel.2019.08.005Google Scholar32Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell SenescenceZhang, Hanlin; Alsaleh, Ghada; Feltham, Jack; Sun, Yizhe; Napolitano, Gennaro; Riffelmacher, Thomas; Charles, Philip; Frau, Lisa; Hublitz, Philip; Yu, Zhanru; Mohammed, Shabaz; Ballabio, Andrea; Balabanov, Stefan; Mellor, Jane; Simon, Anna KatharinaMolecular Cell (2019), 76 (1), 110-125.e9CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central mol. mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.
- 33Mastrodomenico, V., Esin, J. J., Qazi, S., Khomutov, M. A., Ivanov, A. V., Mukhopadhyay, S., and Mounce, B. C. (2020) Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote Entry. ACS Infect. Dis. 6 (9), 2490– 2501, DOI: 10.1021/acsinfecdis.0c00402Google Scholar33Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote EntryMastrodomenico, Vincent; Esin, Jeremy J.; Qazi, Shefah; Khomutov, Maxim A.; Ivanov, Alexander V.; Mukhopadhyay, Suchetana; Mounce, Bryan C.ACS Infectious Diseases (2020), 6 (9), 2490-2501CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these mols. are diverse. One group of cellular metabolites important in virus infection is the polyamines, small pos. charged mols. involved in cell cycle, translation, and nucleic acid metab., among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymic activity. Rift Valley fever virus (RVFV) is a neg. and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-assocd. polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related mols. assoc. with purified virions and transmit from cell to cell during infection. Virion-assocd. spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temp. sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions contg. spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines assocd. with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.
- 34Beigel, J. H., Tomashek, K. M., Dodd, L. E., Mehta, A. K., Zingman, B. S., Kalil, A. C., Hohmann, E., Chu, H. Y., Luetkemeyer, A., Kline, S., Lopez de Castilla, D., Finberg, R. W., Dierberg, K., Tapson, V., Hsieh, L., Patterson, T. F., Paredes, R., Sweeney, D. A., Short, W. R., Touloumi, G., Lye, D. C., Ohmagari, N., Oh, M.-D., Ruiz-Palacios, G. M., Benfield, T., Fätkenheuer, G., Kortepeter, M. G., Atmar, R. L., Creech, C. B., Lundgren, J., Babiker, A. G., Pett, S., Neaton, J. D., Burgess, T. H., Bonnett, T., Green, M., Makowski, M., Osinusi, A., Nayak, S., and Lane, H. C. (2020) ACTT-1 Study Group Members. Remdesivir for the Treatment of Covid-19 - Preliminary Report. N. Engl. J. Med. DOI: 10.1056/NEJMoa2007764Google ScholarThere is no corresponding record for this reference.
- 35Goldman, J. D., Lye, D. C. B., Hui, D. S., Marks, K. M., Bruno, R., Montejano, R., Spinner, C. D., Galli, M., Ahn, M.-Y., Nahass, R. G., Chen, Y.-S., SenGupta, D., Hyland, R. H., Osinusi, A. O., Cao, H., Blair, C., Wei, X., Gaggar, A., Brainard, D. M., Towner, W. J., Muñoz, J., Mullane, K. M., Marty, F. M., Tashima, K. T., Diaz, G., and Subramanian, A. (2020) GS-US-540–5773 Investigators. Remdesivir for 5 or 10 Days in Patients with Severe Covid-19. N. Engl. J. Med. DOI: 10.1056/NEJMoa2015301Google ScholarThere is no corresponding record for this reference.
- 36Grein, J., Ohmagari, N., Shin, D., Diaz, G., Asperges, E., Castagna, A., Feldt, T., Green, G., Green, M. L., Lescure, F.-X., Nicastri, E., Oda, R., Yo, K., Quiros-Roldan, E., Studemeister, A., Redinski, J., Ahmed, S., Bernett, J., Chelliah, D., Chen, D., Chihara, S., Cohen, S. H., Cunningham, J., D’Arminio Monforte, A., Ismail, S., Kato, H., Lapadula, G., L’Her, E., Maeno, T., Majumder, S., Massari, M., Mora-Rillo, M., Mutoh, Y., Nguyen, D., Verweij, E., Zoufaly, A., Osinusi, A. O., DeZure, A., Zhao, Y., Zhong, L., Chokkalingam, A., Elboudwarej, E., Telep, L., Timbs, L., Henne, I., Sellers, S., Cao, H., Tan, S. K., Winterbourne, L., Desai, P., Mera, R., Gaggar, A., Myers, R. P., Brainard, D. M., Childs, R., and Flanigan, T. (2020) Compassionate Use of Remdesivir for Patients with Severe Covid-19. N. Engl. J. Med. 382, 2327, DOI: 10.1056/NEJMoa2007016Google Scholar36Compassionate use of remdesivir for patients with severe Covid-19Grein, J.; Ohmagari, N.; Shin, D.; Diaz, G.; Asperges, E.; Castagna, A.; Feldt, T.; Green, G.; Green, M. L.; Lescure, F.-X.; Nicastri, E.; Oda, R.; Yo, K.; Quiros-Roldan, E.; Studemeister, A.; Redinski, J.; Ahmed, S.; Bernett, J.; Chelliah, D.; Chen, D.; Chihara, S.; Cohen, S. H.; Cunningham, J.; Monforte, A. D'Arminio; Ismail, S.; Kato, H.; Lapadula, G.; L'Her, E.; Maeno, T.; Majumder, S.; Massari, M.; Mora-Rillo, M.; Mutoh, Y.; Nguyen, D.; Verweij, E.; Zoufaly, A.; Osinusi, A. O.; DeZure, A.; Zhao, Y.; Zhong, L.; Chokkalingam, A.; Elboudwarej, E.; Telep, L.; Timbs, L.; Henne, I.; Sellers, S.; Cao, H.; Tan, S. K.; Winterbourne, L.; Desai, P.; Mera, R.; Gaggar, A.; Myers, R. P.; Brainard, D. M.; Childs, R.; Flanigan, T.New England Journal of Medicine (2020), 382 (24), 2327-2336CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)Background: Remdesivir, a nucleotide analog prodrug that inhibits viral RNA polymerases, has shown in vitro activity against SARS-CoV-2. Methods: We provided remdesivir on a compassionate-use basis to patients hospitalized with Covid-19, the illness caused by infection with SARS-CoV-2. Patients were those with confirmed SARS-CoV-2 infection who had an oxygen satn. of 94% or less while they were breathing ambient air or who were receiving oxygen support. Patients received a 10-day course of remdesivir, consisting of 200 mg administered i.v. on day 1, followed by 100 mg daily for the remaining 9 days of treatment. This report is based on data from patients who received remdesivir during the period from Jan. 25, 2020, through March 7, 2020, and have clin. data for at least 1 subsequent day. Results: Of the 61 patients who received at least one dose of remdesivir, data from 8 could not be analyzed (including 7 patients with no post-treatment data and 1 with a dosing error). Of the 53 patients whose data were analyzed, 22 were in the United States, 22 in Europe or Canada, and 9 in Japan. At baseline, 30 patients (57%) were receiving mech. ventilation and 4 (8%) were receiving extracorporeal membrane oxygenation. During a median follow-up of 18 days, 36 patients (68%) had an improvement in oxygen-support class, including 17 of 30 patients (57%) receiving mech. ventilation who were extubated. A total of 25 patients (47%) were discharged, and 7 patients (13%) died; mortality was 18% (6 of 34) among patients receiving invasive ventilation and 5% (1 of 19) among those not receiving invasive ventilation. Conclusions: In this cohort of patients hospitalized for severe Covid-19 who were treated with compassionate-use remdesivir, clin. improvement was obsd. in 36 of 53 patients (68%). Measurement of efficacy will require ongoing randomized, placebo-controlled trials of remdesivir therapy.
- 37Wang, Y., Zhang, D., Du, G., Du, R., Zhao, J., Jin, Y., Fu, S., Gao, L., Cheng, Z., Lu, Q., Hu, Y., Luo, G., Wang, K., Lu, Y., Li, H., Wang, S., Ruan, S., Yang, C., Mei, C., Wang, Y., Ding, D., Wu, F., Tang, X., Ye, X., Ye, Y., Liu, B., Yang, J., Yin, W., Wang, A., Fan, G., Zhou, F., Liu, Z., Gu, X., Xu, J., Shang, L., Zhang, Y., Cao, L., Guo, T., Wan, Y., Qin, H., Jiang, Y., Jaki, T., Hayden, F. G., Horby, P. W., Cao, B., and Wang, C. (2020) Remdesivir in Adults with Severe COVID-19: A Randomised, Double-Blind, Placebo-Controlled, Multicentre Trial. Lancet 395 (10236), 1569– 1578, DOI: 10.1016/S0140-6736(20)31022-9Google Scholar37Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trialWang, Yeming; Zhang, Dingyu; Du, Guanhua; Du, Ronghui; Zhao, Jianping; Jin, Yang; Fu, Shouzhi; Gao, Ling; Cheng, Zhenshun; Lu, Qiaofa; Hu, Yi; Luo, Guangwei; Wang, Ke; Lu, Yang; Li, Huadong; Wang, Shuzhen; Ruan, Shunan; Yang, Chengqing; Mei, Chunlin; Wang, Yi; Ding, Dan; Wu, Feng; Tang, Xin; Ye, Xianzhi; Ye, Yingchun; Liu, Bing; Yang, Jie; Yin, Wen; Wang, Aili; Fan, Guohui; Zhou, Fei; Liu, Zhibo; Gu, Xiaoying; Xu, Jiuyang; Shang, Lianhan; Zhang, Yi; Cao, Lianjun; Guo, Tingting; Wan, Yan; Qin, Hong; Jiang, Yushen; Jaki, Thomas; Hayden, Frederick G.; Horby, Peter W.; Cao, Bin; Wang, ChenLancet (2020), 395 (10236), 1569-1578CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)No specific antiviral drug has been proven effective for treatment of patients with severe coronavirus disease 2019 (COVID-19). Remdesivir (GS-5734), a nucleoside analog prodrug, has inhibitory effects on pathogenic animal and human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, and inhibits Middle East respiratory syndrome coronavirus, SARS-CoV-1, and SARS-CoV-2 replication in animal models. We did a randomized, double-blind, placebo-controlled, multicenter trial at ten hospitals in Hubei, China. Eligible patients were adults (aged ≥18 years) admitted to hospital with lab.-confirmed SARS-CoV-2 infection, with an interval from symptom onset to enrollment of 12 days or less, oxygen satn. of 94% or less on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen of 300 mm Hg or less, and radiol. confirmed pneumonia. Patients were randomly assigned in a 2:1 ratio to i.v. remdesivir (200 mg on day 1 followed by 100 mg on days 2-10 in single daily infusions) or the same vol. of placebo infusions for 10 days. Patients were permitted concomitant use of lopinavir-ritonavir, interferons, and corticosteroids. The primary endpoint was time to clin. improvement up to day 28, defined as the time (in days) from randomization to the point of a decline of two levels on a six-point ordinal scale of clin. status (from 1=discharged to 6=death) or discharged alive from hospital, whichever came first. Primary anal. was done in the intention-to-treat (ITT) population and safety anal. was done in all patients who started their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04257656. Between Feb 6, 2020, and March 12, 2020, 237 patients were enrolled and randomly assigned to a treatment group (158 to remdesivir and 79 to placebo); one patient in the placebo group who withdrew after randomization was not included in the ITT population. Remdesivir use was not assocd. with a difference in time to clin. improvement (hazard ratio 1·23 [95% CI 0·87-1·75]). Although not statistically significant, patients receiving remdesivir had a numerically faster time to clin. improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1·52 [0·95-2·43]). Adverse events were reported in 102 (66%) of 155 remdesivir recipients vs. 50 (64%) of 78 placebo recipients. Remdesivir was stopped early because of adverse events in 18 (12%) patients vs. four (5%) patients who stopped placebo early. In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not assocd. with statistically significant clin. benefits. However, the numerical redn. in time to clin. improvement in those treated earlier requires confirmation in larger studies. Chinese Academy of Medical Sciences Emergency Project of COVID-19, National Key Research and Development Program of China, the Beijing Science and Technol. Project.
- 38Madhubala, R. (1997) Thin-Layer Chromatographic Method for Assaying Polyamines. Methods Mol. Biol. 79, 131– 136, DOI: 10.1385/0-89603-448-8:131Google ScholarThere is no corresponding record for this reference.
- 39Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 9 (7), 671– 675, DOI: 10.1038/nmeth.2089Google Scholar39NIH Image to ImageJ: 25 years of image analysisSchneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W.Nature Methods (2012), 9 (7_part1), 671-675CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the anal. of scientific images. We discuss the origins, challenges and solns. of these two programs, and how their history can serve to advise and inform other software projects.
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Abstract
Figure 1
Figure 1. Polyamine synthesis inhibitor DFMO limits coronavirus replication. (A) Schematic of the polyamine pathway. The polyamines putrescine, spermidine, and spermine are synthesized from ornithine via ornithine decarboxylase (ODC1), which is inhibited by difluoromethylornithine (DFMO). (B) Vero-E6 cells treated with increasing doses of DFMO were infected with SARS-CoV-2 at an MOI of 0.1 for 24 h, and viral genomes were measured and converted to titer equivalents via a standard curve. (C) Vero-E6 cells were treated with escalating DFMO doses and viability and (D) polyamine levels were measured. (E) BHK-R cells were treated with DFMO as indicated and infected with MHV at an MOI of 0.01 for 48 h. (F) Cellular viability levels were measured and (G) polyamine depletion was confirmed by chromatography. (H) BHK-R cells were treated with 1 mM DFMO and infected with MHV at an MOI of 0.01. Viral titers were determined after several rounds of replication. (I) MHV genomes from samples in (E) were quantified by qRT-PCR and compared to viral titers to quantify the relative number of genomes to PFU (J). (K) HeLa-R cells were treated with escalating doses of DFMO and infected with MHV at an MOI of 0.01 for 48 h. (L) HeLa-R cells exposed to DFMO for 4 days were analyzed for viability. (M) Depletion of polyamines was confirmed by chromatography. *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
Figure 2
Figure 2. Polyamines facilitate coronavirus replication. (A) BHK-R cells were treated with escalating doses of putrescine, spermidine, and spermine, and cellular viability was measured 24 h later. Calculated CC50 values are in the inset. (B) BHK-R cells treated as in (A) were infected with MHV at an MOI of 0.01 and titered at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (C) BHK-R cells were treated with 1 mM DFMO for 4 days prior to infection with MHV at an MOI of 0.01. At the time of infection, 1 μM polyamines were added to the cells. Viral titers were determined at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (D) MHV was directly incubated with DFMO or 1 μM of the polyamines putrescine (put), spermidine (spd), and spermine (spm) for 24 h and then directly titered. **p < 0.01 by the Student’s t test. Data from at least two independent experiments.
Figure 3
Figure 3. Polyamine depletion is antiviral prophylactically. Confluent monolayers of BHK-R cells were treated with DFMO at the time of infection with MHV and overlaid with agarose to form plaques. (A) Plaques were quantified 2 days later. (B) Representative plaques are shown from (A). (C) HeLa-R cells were infected and treated as in (A) and the plaque area was measured. (D) BHK-R cells were treated with 1.5 mM DFMO in one, three, or four doses prior to infection with MHV at an MOI of 0.01. Viral titers were determined by plaque assay at 48 hpi. (E) Cells were treated with 1 mM DFMO and then subsequently refed with fresh medium not containing DFMO 24 h before infection with MHV at an MOI of 0.01. Titers were determined at 24 hpi. **p < 0.01 and ***p < 0.001 by the Student’s t test. Representative data from at least three independent experiments. Individual plaque sizes are indicated in (A) and (C).
Figure 4
Figure 4. Polyamines facilitate coronavirus cellular attachment. Confluent BHK-R cells were treated with DFMO prior to the MHV binding assay. Virus was inoculated on cells for 5 min prior to removal, wash, and agarose overlay in media without DFMO. Plaques formed were quantified (A), and representative images from 1 mM DFMO treatment are shown (B). (C) BHK-R cells were treated as in (A), but after washing unattached virus, attached virus and cells were collected for RNA purification, reverse transcription, and viral genome quantitation by qPCR. Attached genomes were normalized to input virus. (D) MHV receptor (CECAM-1) was visualized on untreated and 1 mM DFMO treated cells. (E) HeLa-R cells were treated with 1 mM DFMO, infected with MHV, and processed as in (C). (F) Vero-E6 cells were treated with DFMO and analyzed for binding ability of HCoV-NL63 as in (C). (G) ACE2 expression on Vero-E6 cells was confirmed by immunofluorescence. (H) Pseudotyped viral particles with no viral glycoproteins or the SARS-CoV-2 spike were tested for their ability to bind and enter Vero-E6 cells. (I) Spike protein was confirmed to be present on pseudotyped virus by western blot. (J) Vero-E6 cells were treated with DFMO and infected with SARS-CoV-2 pseudoparticles as in (C). *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
Figure 5
Figure 5. Additional molecules targeting the polyamine pathway quell coronavirus infection. (A) Schematic of the polyamine and hypusination pathway. (B) HeLa-R cells were treated with escalating doses of DENSpm for 16 h prior to infection with MHV at an MOI of 0.01. Viral titers were determined at 48 hpi (upper). Polyamine levels were measured by chromatography (lower). (C) Treated cells were assayed for viability after 16 h of treatment with DENSpm. (D) HeLa-R cells were treated with DENSpm at the indicated times before and after infection with MHV at an MOI of 10. Viral titers were determined at 48 hpi. BHK-R cells were treated with (E) CPX, (F) DEF, and (G) GC7 for 24 h prior to infection with MHV at an MOI of 0.01. Viral titers were determined at 48 hpi. (H) BHK-R cells treated with CPX, DEF, and GC7 were analyzed for cell viability after 24 h of treatment. *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t test. Data from at least three independent experiments.
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- 5van der Hoek, L., Ihorst, G., Sure, K., Vabret, A., Dijkman, R., de Vries, M., Forster, J., Berkhout, B., and Uberla, K. (2010) Burden of Disease Due to Human Coronavirus NL63 Infections and Periodicity of Infection. J. Clin. Virol. 48 (2), 104– 108, DOI: 10.1016/j.jcv.2010.02.0235Burden of disease due to human coronavirus NL63 infections and periodicity of infectionvan der Hoek Lia; Ihorst Gabriele; Sure Klaus; Vabret Astrid; Dijkman Ronald; de Vries Michel; Forster Johannes; Berkhout Ben; Uberla KlausJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology (2010), 48 (2), 104-8 ISSN:.BACKGROUND: The disease burden caused by recently identified respiratory viruses like HCoV-NL63 is unknown. OBJECTIVES: We determined the burden of disease due to HCoV-NL63 infections using the population-based PRI.DE cohort of children under the age of 3 with lower respiratory tract infections (LRTIs). STUDY DESIGN: In total 1756 respiratory samples, from hospitalized children or children who visited the outpatient clinic, were tested for HCoV-NL63. Sampling covered a period of 2 years and the frequency of infection in different years was compared to other Western European studies that tested for this virus in 2 or more consecutive years. RESULTS: Sixty-nine samples were HCoV-NL63 positive, 35 were with high loads, and of these 25 were single HCoV-NL63 infections. Based on the number of children with high HCoV-NL63 infection and no additional infection, the overall annual incidence in outpatients was 7 per 1000 children per year (95% confidence interval (CI) 3-13 per 1000 children per year), which can be extrapolated to an absolute number of 16,929 visits to the physician due to an HCoV-NL63 infection in Germany per year. The estimated hospitalization rate is 22 per 100,000 children (95% CI: 7-49 per 100,000 children per year). This number reflects 522 HCoV-NL63 children in Germany per year. A large year-to-year difference in HCoV-NL63 infection frequency was observed. Combining these data with those of other studies in Western Europe revealed that HCoV-NL63 infections follow a 2-year inter-epidemic period with peaks of infection in the winters of 2000/2001, 2002/2003 and 2004/2005 (p<0.0001). CONCLUSIONS: HCoV-NL63 infection in children below 3 years of age often requires a visit to the physician in an outpatient clinic, especially during peak-years, but hospitalizations are relatively infrequent.
- 6Brown, A. J., Won, J. J., Graham, R. L., Dinnon, K. H., Sims, A. C., Feng, J. Y., Cihlar, T., Denison, M. R., Baric, R. S., and Sheahan, T. P. (2019) Broad Spectrum Antiviral Remdesivir Inhibits Human Endemic and Zoonotic Deltacoronaviruses with a Highly Divergent RNA Dependent RNA Polymerase. Antiviral Res. 169, 104541, DOI: 10.1016/j.antiviral.2019.1045416Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymeraseBrown, Ariane J.; Won, John J.; Graham, Rachel L.; Dinnon, Kenneth H., III; Sims, Amy C.; Feng, Joy Y.; Cihlar, Tomas; Denison, Mark R.; Baric, Ralph S.; Sheahan, Timothy P.Antiviral Research (2019), 169 (), 104541CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV.
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- 8Gerner, E. W. and Meyskens, F. L. (2004) Polyamines and Cancer: Old Molecules, New Understanding. Nat. Rev. Cancer 4 (10), 781– 792, DOI: 10.1038/nrc14548Polyamines and cancer: Old molecules, new understandingGerner, Eugene W.; Meyskens, Frank L., Jr.Nature Reviews Cancer (2004), 4 (10), 781-792CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)A review. The amino-acid-derived polyamines have long been assocd. with cell growth and cancer, and specific oncogenes and tumor-suppressor genes regulate polyamine metab. Inhibition of polyamines synthesis has proven to be generally ineffective as an anticancer strategy in clin. trials, but it is a potent cancer chemoprevention strategy in preclin. studies. Clin. trials, with well-defined goals, are now underway to evaluate the chemopreventive efficacy of inhibitors of polyamine synthesis in a range of tissues.
- 9Frugier, M., Florentz, C., Hosseini, M. W., Lehn, J. M., and Giegé, R. (1994) Synthetic Polyamines Stimulate in Vitro Transcription by T7 RNA Polymerase. Nucleic Acids Res. 22 (14), 2784– 2790, DOI: 10.1093/nar/22.14.27849Synthetic polyamines stimulate in vitro transcription by T7 RNA polymeraseFrugier, Magali; Florentz, Catherine; Hosseini, Mir Wais; Lehn, Jean Marie; Giege, RichardNucleic Acids Research (1994), 22 (14), 2784-90CODEN: NARHAD; ISSN:0305-1048.The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compds. used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and no. of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulated transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concn. For each compd., an optimal concn. could be defined; above this concn., transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compd. called [38]N6C10.
- 10Mandal, S., Mandal, A., Johansson, H. E., Orjalo, A. V., and Park, M. H. (2013) Depletion of Cellular Polyamines, Spermidine and Spermine, Causes a Total Arrest in Translation and Growth in Mammalian Cells. Proc. Natl. Acad. Sci. U. S. A. 110 (6), 2169– 2174, DOI: 10.1073/pnas.121900211010Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cellsMandal, Swati; Mandal, Ajeet; Johansson, Hans E.; Orjalo, Arturo V.; Park, Myung HeeProceedings of the National Academy of Sciences of the United States of America (2013), 110 (6), 2169-2174, S2169/1-S2169/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromol. synthesis in vitro, their specific functions in vivo are poorly understood. Here, the authors investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N1-acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with green fluorescent protein (GFP) and SAT1 vectors suppressed GFP expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect was most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggested a direct role for polyamines in translation initiation. The data provided strong evidence for a primary function of the polyamines, spermidine and spermine, in translation in mammalian cells.
- 11Burri, C. and Brun, R. (2003) Eflornithine for the Treatment of Human African Trypanosomiasis. Parasitol. Res. 90 (S1), S49– 52, DOI: 10.1007/s00436-002-0766-5There is no corresponding record for this reference.
- 12Milord, F., Pépin, J., Loko, L., Ethier, L., and Mpia, B. (1992) Efficacy and Toxicity of Eflornithine for Treatment of Trypanosoma Brucei Gambiense Sleeping Sickness. Lancet 340 (8820), 652– 655, DOI: 10.1016/0140-6736(92)92180-N12Efficacy and toxicity of eflornithine for treatment of Trypanosoma brucei gambiense sleeping sicknessMilord F; Pepin J; Loko L; Ethier L; Mpia BLancet (London, England) (1992), 340 (8820), 652-5 ISSN:0140-6736.The usual first-line treatment for Trypanosoma brucei gambiense sleeping sickness is melarsoprol, but when that fails the outlook has hitherto been grim. The polyamine synthesis inhibitor eflornithine (difluoromethylornithine, DFMO) has emerged as an alternative therapy. 207 patients with late-stage T b gambiense sleeping sickness were treated in rural Zaire with three different regimens of DFMO in an open-trial design. During treatment, trypanosomes disappeared from the CSF of all 87 patients in whom parasites had been seen before DFMO administration, and there was a sharp fall in CSF white cell count from a mean of 186/microliters to 21/microliters. 152 patients have been followed for at least a year after DFMO treatment, and only 13 (9%) have relapsed. Treatment failures were more common in children less than 12 years, among patients treated with oral DFMO only, and among patients who received DFMO as the initial treatment of their recently diagnosed trypanosomiasis. Toxicity was acceptable. Only 4 patients died during or shortly after treatment. Bone marrow suppression resulting in anaemia (43%) or leucopenia (53%) was common but bore little consequence. This open trial shows that DFMO is as active as and possibly less toxic than melarsoprol. For economic and logistic reasons DFMO may not be the first-choice therapy in rural Africa but for the vast majority of patients who relapse after melarsoprol DFMO will be curative.
- 13Shapiro, J. and Lui, H. (2001) Vaniqa - Eflornithine 13.9% Cream. Skin Therapy Letter 6 (7), 113Vaniqa--eflornithine 13.9% creamShapiro J; Lui HSkin therapy letter (2001), 6 (7), 1-3, 5 ISSN:1201-5989.Eflornithine HCl 13.9% cream is the first topical prescription treatment to be approved by the US FDA for the reduction of unwanted facial hair in women. It irreversibly inhibits ornithine decarboxylase (ODC), an enzyme that catalyzes the rate-limiting step for follicular polyamine synthesis, which is necessary for hair growth. In clinical trials eflornithine cream slowed the growth of unwanted facial hair in up to 60% of women. Improvement occurs gradually over a period of 4-8 weeks or longer. Most reported adverse reactions consisted of minor skin irritation.
- 14Mounce, B. C., Poirier, E. Z., Passoni, G., Simon-Loriere, E., Cesaro, T., Prot, M., Stapleford, K. A., Moratorio, G., Sakuntabhai, A., Levraud, J.-P., and Vignuzzi, M. (2016) Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses. Cell Host Microbe 20 (2), 167– 177, DOI: 10.1016/j.chom.2016.06.01114Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya VirusesMounce, Bryan C.; Poirier, Enzo Z.; Passoni, Gabriella; Simon-Loriere, Etienne; Cesaro, Teresa; Prot, Matthieu; Stapleford, Kenneth A.; Moratorio, Gonzalo; Sakuntabhai, Anavaj; Levraud, Jean-Pierre; Vignuzzi, MarcoCell Host & Microbe (2016), 20 (2), 167-177CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Polyamines are small, pos. charged mols. derived from ornithine and synthesized through an intricately regulated enzymic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these mols. in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies.
- 15Kicmal, T. M., Tate, P. M., Dial, C. N., Esin, J. J., and Mounce, B. C. (2019) Polyamine Depletion Abrogates Enterovirus Cellular Attachment. J. Virol. 93, e01054-19, DOI: 10.1128/JVI.01054-19There is no corresponding record for this reference.
- 16Olsen, M. E., Filone, C. M., Rozelle, D., Mire, C. E., Agans, K. N., Hensley, L., and Connor, J. H. (2016) Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication. mBio 7 (4), e00882-16, DOI: 10.1128/mBio.00882-16There is no corresponding record for this reference.
- 17Mounce, B. C., Olsen, M. E., Vignuzzi, M., and Connor, J. H. (2017) Polyamines and Their Role in Virus Infection. Microbiol. Mol. Biol. Rev. 81 (4), e00029-17, DOI: 10.1128/MMBR.00029-17There is no corresponding record for this reference.
- 18Firpo, M. R. and Mounce, B. C. (2020) Diverse Functions of Polyamines in Virus Infection. Biomolecules 10 (4), 628, DOI: 10.3390/biom1004062818Diverse functions of polyamines in virus infectionFirpo, Mason R.; Mounce, Bryan C.Biomolecules (2020), 10 (4), 628CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)A review. As obligate intracellular parasites, viruses rely on host cells for the building blocks of progeny viruses. Metabolites such as amino acids, nucleotides and lipids are central to viral proteins, genomes, and envelopes, and the availability of these mols. can restrict or promote infection. Polyamines, comprised of putrescine, spermidine, and spermine in mammalian cells, are also crit. for virus infection. Polyamines are small, pos. charged mols. that function in transcription, translation, and cell cycling. Initial work on the function of polyamines in bacteriophage infection illuminated these mols. as crit. to virus infection. In the decades since early virus-polyamine descriptions, work on diverse viruses continues to highlight a role for polyamines in viral processes, including genome packaging and viral enzymic activity. On the host side, polyamines function in the response to virus infection. Thus, viruses and hosts compete for polyamines, which are a crit. resource for both. Pharmacol. targeting polyamines, tipping the balance to favor the host and restrict virus replication, holds significant promise as a broad-spectrum antiviral strategy.
- 19Mounce, B. C., Cesaro, T., Moratorio, G., Hooikaas, P. J., Yakovleva, A., Werneke, S. W., Smith, E. C., Poirier, E. Z., Simon-Loriere, E., Prot, M., Tamietti, C., Vitry, S., Volle, R., Khou, C., Frenkiel, M.-P., Sakuntabhai, A., Delpeyroux, F., Pardigon, N., Flamand, M., Barba-Spaeth, G., Lafon, M., Denison, M. R., Albert, M. L., and Vignuzzi, M. (2016) Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. J. Virol. 90 (21), 9683– 9692, DOI: 10.1128/JVI.01347-1619Inhibition of polyamine biosynthesis is a broad-spectrum strategy against RNA virusesMounce, Bryan C.; Cesaro, Teresa; Moratorio, Gonzalo; Hooikaas, Peter Jan; Yakovleva, Anna; Werneke, Scott W.; Smith, Everett Clinton; Poirier, Enzo Z.; Simon-Loriere, Etienne; Prot, Matthieu; Tamietti, Carole; Vitry, Sandrine; Volle, Romain; Khou, Cecile; Frenkiel, Marie-Pascale; Sakuntabhai, Anavaj; Delpeyroux, Francis; Pardigon, Nathalie; Flamand, Marie; Barba-Spaeth, Giovanna; Lafon, Monique; Denison, Mark R.; Albert, Matthew L.; Vignuzzi, MarcoJournal of Virology (2016), 90 (21), 9683-9692CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compds. show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant pos. charged mols. found in the cell. Here, we describe the antiviral effects of two mols. that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a neg. effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo. These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available mols., such as DFMO.
- 20Compton, S. R., Barthold, S. W., and Smith, A. L. (1993) The Cellular and Molecular Pathogenesis of Coronaviruses. Lab. Anim. Sci. 43 (1), 15– 2820The cellular and molecular pathogenesis of coronavirusesCompton S R; Barthold S W; Smith A LLaboratory animal science (1993), 43 (1), 15-28 ISSN:0023-6764.Coronaviruses cause a wide spectrum of diseases in humans and animals but generally fall into two classes, with respiratory or enteric tropisms. Mouse hepatitis virus (MHV) and rat coronaviruses are the viruses most frequently encountered in the laboratory animal setting. This review focuses primarily on the cellular and molecular aspects of MHV pathogenesis. The high mutation and recombination rates of coronaviruses lead to a diverse, ever-changing population of MHV strains. The spike (S) protein is the most variable coronavirus protein and is responsible for binding to cell surface receptors, inducing cell fusion and humoral and cellular immunity. Differences within the S protein of different MHV strains have been linked to their variable tropisms. Since immunity to MHV is strain-specific, seropositive mice can be reinfected with different strains of MHV. Natural infections with MHV are acute, with persistence occurring at the population level, not within an individual mouse, unless it is immunocompromised. Age, genotype, immunologic status of the mouse, and MHV strain influence the type and severity of disease caused by MHV. Interference with research by MHV has been reported primarily in the fields of immunology and tumor biology and may be a reflection of MHV's capacity to grow in several types of immune cells. While many methods are available to diagnose coronavirus infection, serologic tests, primarily ELISA and IFA, are the most commonly used. MHV is best managed on a preventive basis. Elimination of MHV from a population requires cessation of breeding and halting the introduction of naive mice into the population.
- 21Weiss, S. R., and Leibowitz, J. L. Coronavirus Pathogenesis. In Advances in Virus Research; Maramorosch, K., Shatkin, A. J., and Murphy, F. A., Eds.; Academic Press, 2011; Vol. 81, Chapter 4, pp 85– 164; DOI: 10.1016/B978-0-12-385885-6.00009-2 .There is no corresponding record for this reference.
- 22Leibowitz, J., Kaufman, G., and Liu, P. (2011) Coronaviruses: Propagation, Quantification, Storage, and Construction of Recombinant Mouse Hepatitis Virus. Curr. Protoc. Microbiol. 21, 15E.1.1– 15E.1.46, DOI: 10.1002/9780471729259.mc15e01s21There is no corresponding record for this reference.
- 23Mastrodomenico, V., Esin, J. J., Graham, M. L., Tate, P. M., Hawkins, G. M., Sandler, Z. J., Rademacher, D. J., Kicmal, T. M., Dial, C. N., and Mounce, B. C. (2019) Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions. J. Virol. 93, e00530-19, DOI: 10.1128/JVI.00530-19There is no corresponding record for this reference.
- 24Gassen, N. C., Papies, J., Bajaj, T., Dethloff, F., Emanuel, J., Weckmann, K., Heinz, D. E., Heinemann, N., Lennarz, M., Richter, A., Niemeyer, D., Corman, V. M., Giavalisco, P., Drosten, C., and Müller, M. A. (2020) Analysis of SARS-CoV-2-Controlled Autophagy Reveals Spermidine, MK-2206, and Niclosamide as Putative Antiviral Therapeutics. bioRxiv, 2020.04.15.997254, DOI: 10.1101/2020.04.15.997254 .There is no corresponding record for this reference.
- 25Igarashi, K. and Kashiwagi, K. (2000) Polyamines: Mysterious Modulators of Cellular Functions. Biochem. Biophys. Res. Commun. 271 (3), 559– 564, DOI: 10.1006/bbrc.2000.260125Polyamines: Mysterious Modulators of Cellular FunctionsIgarashi, Kazuei; Kashiwagi, KeikoBiochemical and Biophysical Research Communications (2000), 271 (3), 559-564CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)A review, with ∼67 refs. In recent years the functions of polyamines (putrescine, spermidine, and spermine) have been studied at the mol. level. Polyamines can modulate the functions of RNA, DNA, nucleotide triphosphates, proteins, and other acidic substances. A major part of the cellular functions of polyamines can be explained through a structural change of RNA which occurs at physiol. concns. of Mg2+ and K+ because most polyamines exist in a polyamine-RNA complex within cells. Polyamines were found to modulate protein synthesis at several different levels including stimulation of special kinds of protein synthesis, stimulation of the assembly of 30 S ribosomal subunits and stimulation of Ile-tRNA formation. Effects of polyamines on ion channels have also been reported and are gradually being clarified at the mol. level. (c) 2000 Academic Press.
- 26Igarashi, K. and Kashiwagi, K. (2019) The Functional Role of Polyamines in Eukaryotic Cells. Int. J. Biochem. Cell Biol. 107, 104– 115, DOI: 10.1016/j.biocel.2018.12.012There is no corresponding record for this reference.
- 27Mastrodomenico, V., Esin, J. J., Qazi, S., Omoba, O. S., Fung, B. L., Khomutov, M. A., Ivanov, A. V., Mukhopadhyay, S., and Mounce, B. C. (2020) Virion-Associated Spermidine Transmits with Rift Valley Fever Virus Particles to Maintain Infectivity. bioRxiv, 2020.01.23.915900, DOI: 10.1101/2020.01.23.915900 .There is no corresponding record for this reference.
- 28Wolf, J. E., Shander, D., Huber, F., Jackson, J., Lin, C.-S., Mathes, B. M., and Schrode, K. (2007) Eflornithine HCl Study Group. Randomized, Double-Blind Clinical Evaluation of the Efficacy and Safety of Topical Eflornithine HCl 13.9% Cream in the Treatment of Women with Facial Hair. Int. J. Dermatol. 46 (1), 94– 98, DOI: 10.1111/j.1365-4632.2006.03079.x28Randomized, double-blind clinical evaluation of the efficacy and safety of topical eflornithine HCl 13.9% cream in the treatment of women with facial hairWolf, John E., Jr.; Shander, Douglas; Huber, Ferdinand; Jackson, Joseph; Lin, Chen-Sheng; Mathes, Barbara M.; Schrode, Kathy; Bergfeld, Wilma F.; Camacho, Francisco; Dobs, Adrian S.; Dunlap, Frank; Hordinsky, Maria; Katz, Irving H.; Lebwohl, Mark; McMichael, Amy; Olsen, Elise A.; Pariser, David M.; Piacquadio, Daniel; Price, Vera; Redmond, Geoffrey P.; Rodriguez, David; Sawaya, Marty E.; Weiss, Jonathan S.; Whiting, David A.; Wilson, David C.; Wolf, John E., Jr.International Journal of Dermatology (2007), 46 (1), 94-98CODEN: IJDEBB; ISSN:0011-9059. (Blackwell Publishing Ltd.)Geater than 40% of women in the USA have unwanted facial hair, a condition that can have a profound impact on a woman's self-esteem, confidence, and overall quality-of-life. Eflornithine is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting enzyme in the synthesis of polyamines, which appears to be crit. to the proliferation of matrix cells in the hair follicle and hence to hair growth. This paper reports the results of two double-blind studies evaluating the efficacy and safety of eflornithine HCl 13.9% cream in women with unwanted facial hair. Eflornithine cream, applied twice daily for 24 wk was well tolerated and significantly reduced the growth of unwanted facial hair as measured by a physician's assessment and hair length/mass. Efficacy was not dependent on the method of hair removal. The treatment was generally well tolerated with some patients experiencing mild, transient burning, stinging, or tingling.
- 29Qing, E., Hantak, M., Perlman, S., and Gallagher, T. (2020) Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection. mBio 11 (1), e02764-19 DOI: 10.1128/mBio.02764-19There is no corresponding record for this reference.
- 30Park, M. H. and Wolff, E. C. (2018) Hypusine, a Polyamine-Derived Amino Acid Critical for Eukaryotic Translation. J. Biol. Chem. 293 (48), 18710– 18718, DOI: 10.1074/jbc.TM118.00334130Hypusine, a polyamine-derived amino acid critical for eukaryotic translationPark, Myung Hee; Wolff, Edith C.Journal of Biological Chemistry (2018), 293 (48), 18710-18718CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)A review. The natural amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) is derived from the polyamine spermidine, and occurs only in a single family of cellular proteins, eukaryotic translation factor 5A (eIF5A) isoforms. Hypusine is formed by conjugation of the aminobutyl moiety of spermidine to a specific lysine residue of this protein. The post-translational synthesis of hypusine involves two enzymic steps, catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for eIF5A activity. Inactivation of either the eIF5A or the DHPS gene is lethal in yeast and mouse, underscoring the vital role of eIF5A hypusination in eukaryotic cell growth and animal development. The long and basic side chain of the hypusine residue promotes eIF5A-mediated translation elongation by facilitating peptide bond formation at polyproline stretches and at many other ribosome-pausing sites. It also enhances translation termination by stimulating peptide release. By promoting translation, the hypusine modification of eIF5A provides a key link between polyamines and cell growth regulation. eIF5A has been implicated in several human pathol. conditions. Recent genetic data suggest that eIF5A haploinsufficiency or impaired deoxyhypusine synthase activity is assocd. with neurodevelopmental disorders in humans.
- 31Olsen, M. E., Cressey, T. N., Mühlberger, E., and Connor, J. H. (2018) Differential Mechanisms for the Involvement of Polyamines and Hypusinated EIF5A in Ebola Virus Gene Expression. J. Virol. 92, e01260-18, DOI: 10.1128/JVI.01260-18There is no corresponding record for this reference.
- 32Zhang, H., Alsaleh, G., Feltham, J., Sun, Y., Napolitano, G., Riffelmacher, T., Charles, P., Frau, L., Hublitz, P., Yu, Z., Mohammed, S., Ballabio, A., Balabanov, S., Mellor, J., and Simon, A. K. (2019) Polyamines Control EIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence. Mol. Cell 76 (1), 110– 125.E9, DOI: 10.1016/j.molcel.2019.08.00532Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell SenescenceZhang, Hanlin; Alsaleh, Ghada; Feltham, Jack; Sun, Yizhe; Napolitano, Gennaro; Riffelmacher, Thomas; Charles, Philip; Frau, Lisa; Hublitz, Philip; Yu, Zhanru; Mohammed, Shabaz; Ballabio, Andrea; Balabanov, Stefan; Mellor, Jane; Simon, Anna KatharinaMolecular Cell (2019), 76 (1), 110-125.e9CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central mol. mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.
- 33Mastrodomenico, V., Esin, J. J., Qazi, S., Khomutov, M. A., Ivanov, A. V., Mukhopadhyay, S., and Mounce, B. C. (2020) Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote Entry. ACS Infect. Dis. 6 (9), 2490– 2501, DOI: 10.1021/acsinfecdis.0c0040233Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote EntryMastrodomenico, Vincent; Esin, Jeremy J.; Qazi, Shefah; Khomutov, Maxim A.; Ivanov, Alexander V.; Mukhopadhyay, Suchetana; Mounce, Bryan C.ACS Infectious Diseases (2020), 6 (9), 2490-2501CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these mols. are diverse. One group of cellular metabolites important in virus infection is the polyamines, small pos. charged mols. involved in cell cycle, translation, and nucleic acid metab., among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymic activity. Rift Valley fever virus (RVFV) is a neg. and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-assocd. polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related mols. assoc. with purified virions and transmit from cell to cell during infection. Virion-assocd. spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temp. sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions contg. spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines assocd. with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.
- 34Beigel, J. H., Tomashek, K. M., Dodd, L. E., Mehta, A. K., Zingman, B. S., Kalil, A. C., Hohmann, E., Chu, H. Y., Luetkemeyer, A., Kline, S., Lopez de Castilla, D., Finberg, R. W., Dierberg, K., Tapson, V., Hsieh, L., Patterson, T. F., Paredes, R., Sweeney, D. A., Short, W. R., Touloumi, G., Lye, D. C., Ohmagari, N., Oh, M.-D., Ruiz-Palacios, G. M., Benfield, T., Fätkenheuer, G., Kortepeter, M. G., Atmar, R. L., Creech, C. B., Lundgren, J., Babiker, A. G., Pett, S., Neaton, J. D., Burgess, T. H., Bonnett, T., Green, M., Makowski, M., Osinusi, A., Nayak, S., and Lane, H. C. (2020) ACTT-1 Study Group Members. Remdesivir for the Treatment of Covid-19 - Preliminary Report. N. Engl. J. Med. DOI: 10.1056/NEJMoa2007764There is no corresponding record for this reference.
- 35Goldman, J. D., Lye, D. C. B., Hui, D. S., Marks, K. M., Bruno, R., Montejano, R., Spinner, C. D., Galli, M., Ahn, M.-Y., Nahass, R. G., Chen, Y.-S., SenGupta, D., Hyland, R. H., Osinusi, A. O., Cao, H., Blair, C., Wei, X., Gaggar, A., Brainard, D. M., Towner, W. J., Muñoz, J., Mullane, K. M., Marty, F. M., Tashima, K. T., Diaz, G., and Subramanian, A. (2020) GS-US-540–5773 Investigators. Remdesivir for 5 or 10 Days in Patients with Severe Covid-19. N. Engl. J. Med. DOI: 10.1056/NEJMoa2015301There is no corresponding record for this reference.
- 36Grein, J., Ohmagari, N., Shin, D., Diaz, G., Asperges, E., Castagna, A., Feldt, T., Green, G., Green, M. L., Lescure, F.-X., Nicastri, E., Oda, R., Yo, K., Quiros-Roldan, E., Studemeister, A., Redinski, J., Ahmed, S., Bernett, J., Chelliah, D., Chen, D., Chihara, S., Cohen, S. H., Cunningham, J., D’Arminio Monforte, A., Ismail, S., Kato, H., Lapadula, G., L’Her, E., Maeno, T., Majumder, S., Massari, M., Mora-Rillo, M., Mutoh, Y., Nguyen, D., Verweij, E., Zoufaly, A., Osinusi, A. O., DeZure, A., Zhao, Y., Zhong, L., Chokkalingam, A., Elboudwarej, E., Telep, L., Timbs, L., Henne, I., Sellers, S., Cao, H., Tan, S. K., Winterbourne, L., Desai, P., Mera, R., Gaggar, A., Myers, R. P., Brainard, D. M., Childs, R., and Flanigan, T. (2020) Compassionate Use of Remdesivir for Patients with Severe Covid-19. N. Engl. J. Med. 382, 2327, DOI: 10.1056/NEJMoa200701636Compassionate use of remdesivir for patients with severe Covid-19Grein, J.; Ohmagari, N.; Shin, D.; Diaz, G.; Asperges, E.; Castagna, A.; Feldt, T.; Green, G.; Green, M. L.; Lescure, F.-X.; Nicastri, E.; Oda, R.; Yo, K.; Quiros-Roldan, E.; Studemeister, A.; Redinski, J.; Ahmed, S.; Bernett, J.; Chelliah, D.; Chen, D.; Chihara, S.; Cohen, S. H.; Cunningham, J.; Monforte, A. D'Arminio; Ismail, S.; Kato, H.; Lapadula, G.; L'Her, E.; Maeno, T.; Majumder, S.; Massari, M.; Mora-Rillo, M.; Mutoh, Y.; Nguyen, D.; Verweij, E.; Zoufaly, A.; Osinusi, A. O.; DeZure, A.; Zhao, Y.; Zhong, L.; Chokkalingam, A.; Elboudwarej, E.; Telep, L.; Timbs, L.; Henne, I.; Sellers, S.; Cao, H.; Tan, S. K.; Winterbourne, L.; Desai, P.; Mera, R.; Gaggar, A.; Myers, R. P.; Brainard, D. M.; Childs, R.; Flanigan, T.New England Journal of Medicine (2020), 382 (24), 2327-2336CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)Background: Remdesivir, a nucleotide analog prodrug that inhibits viral RNA polymerases, has shown in vitro activity against SARS-CoV-2. Methods: We provided remdesivir on a compassionate-use basis to patients hospitalized with Covid-19, the illness caused by infection with SARS-CoV-2. Patients were those with confirmed SARS-CoV-2 infection who had an oxygen satn. of 94% or less while they were breathing ambient air or who were receiving oxygen support. Patients received a 10-day course of remdesivir, consisting of 200 mg administered i.v. on day 1, followed by 100 mg daily for the remaining 9 days of treatment. This report is based on data from patients who received remdesivir during the period from Jan. 25, 2020, through March 7, 2020, and have clin. data for at least 1 subsequent day. Results: Of the 61 patients who received at least one dose of remdesivir, data from 8 could not be analyzed (including 7 patients with no post-treatment data and 1 with a dosing error). Of the 53 patients whose data were analyzed, 22 were in the United States, 22 in Europe or Canada, and 9 in Japan. At baseline, 30 patients (57%) were receiving mech. ventilation and 4 (8%) were receiving extracorporeal membrane oxygenation. During a median follow-up of 18 days, 36 patients (68%) had an improvement in oxygen-support class, including 17 of 30 patients (57%) receiving mech. ventilation who were extubated. A total of 25 patients (47%) were discharged, and 7 patients (13%) died; mortality was 18% (6 of 34) among patients receiving invasive ventilation and 5% (1 of 19) among those not receiving invasive ventilation. Conclusions: In this cohort of patients hospitalized for severe Covid-19 who were treated with compassionate-use remdesivir, clin. improvement was obsd. in 36 of 53 patients (68%). Measurement of efficacy will require ongoing randomized, placebo-controlled trials of remdesivir therapy.
- 37Wang, Y., Zhang, D., Du, G., Du, R., Zhao, J., Jin, Y., Fu, S., Gao, L., Cheng, Z., Lu, Q., Hu, Y., Luo, G., Wang, K., Lu, Y., Li, H., Wang, S., Ruan, S., Yang, C., Mei, C., Wang, Y., Ding, D., Wu, F., Tang, X., Ye, X., Ye, Y., Liu, B., Yang, J., Yin, W., Wang, A., Fan, G., Zhou, F., Liu, Z., Gu, X., Xu, J., Shang, L., Zhang, Y., Cao, L., Guo, T., Wan, Y., Qin, H., Jiang, Y., Jaki, T., Hayden, F. G., Horby, P. W., Cao, B., and Wang, C. (2020) Remdesivir in Adults with Severe COVID-19: A Randomised, Double-Blind, Placebo-Controlled, Multicentre Trial. Lancet 395 (10236), 1569– 1578, DOI: 10.1016/S0140-6736(20)31022-937Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trialWang, Yeming; Zhang, Dingyu; Du, Guanhua; Du, Ronghui; Zhao, Jianping; Jin, Yang; Fu, Shouzhi; Gao, Ling; Cheng, Zhenshun; Lu, Qiaofa; Hu, Yi; Luo, Guangwei; Wang, Ke; Lu, Yang; Li, Huadong; Wang, Shuzhen; Ruan, Shunan; Yang, Chengqing; Mei, Chunlin; Wang, Yi; Ding, Dan; Wu, Feng; Tang, Xin; Ye, Xianzhi; Ye, Yingchun; Liu, Bing; Yang, Jie; Yin, Wen; Wang, Aili; Fan, Guohui; Zhou, Fei; Liu, Zhibo; Gu, Xiaoying; Xu, Jiuyang; Shang, Lianhan; Zhang, Yi; Cao, Lianjun; Guo, Tingting; Wan, Yan; Qin, Hong; Jiang, Yushen; Jaki, Thomas; Hayden, Frederick G.; Horby, Peter W.; Cao, Bin; Wang, ChenLancet (2020), 395 (10236), 1569-1578CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)No specific antiviral drug has been proven effective for treatment of patients with severe coronavirus disease 2019 (COVID-19). Remdesivir (GS-5734), a nucleoside analog prodrug, has inhibitory effects on pathogenic animal and human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, and inhibits Middle East respiratory syndrome coronavirus, SARS-CoV-1, and SARS-CoV-2 replication in animal models. We did a randomized, double-blind, placebo-controlled, multicenter trial at ten hospitals in Hubei, China. Eligible patients were adults (aged ≥18 years) admitted to hospital with lab.-confirmed SARS-CoV-2 infection, with an interval from symptom onset to enrollment of 12 days or less, oxygen satn. of 94% or less on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen of 300 mm Hg or less, and radiol. confirmed pneumonia. Patients were randomly assigned in a 2:1 ratio to i.v. remdesivir (200 mg on day 1 followed by 100 mg on days 2-10 in single daily infusions) or the same vol. of placebo infusions for 10 days. Patients were permitted concomitant use of lopinavir-ritonavir, interferons, and corticosteroids. The primary endpoint was time to clin. improvement up to day 28, defined as the time (in days) from randomization to the point of a decline of two levels on a six-point ordinal scale of clin. status (from 1=discharged to 6=death) or discharged alive from hospital, whichever came first. Primary anal. was done in the intention-to-treat (ITT) population and safety anal. was done in all patients who started their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04257656. Between Feb 6, 2020, and March 12, 2020, 237 patients were enrolled and randomly assigned to a treatment group (158 to remdesivir and 79 to placebo); one patient in the placebo group who withdrew after randomization was not included in the ITT population. Remdesivir use was not assocd. with a difference in time to clin. improvement (hazard ratio 1·23 [95% CI 0·87-1·75]). Although not statistically significant, patients receiving remdesivir had a numerically faster time to clin. improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1·52 [0·95-2·43]). Adverse events were reported in 102 (66%) of 155 remdesivir recipients vs. 50 (64%) of 78 placebo recipients. Remdesivir was stopped early because of adverse events in 18 (12%) patients vs. four (5%) patients who stopped placebo early. In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not assocd. with statistically significant clin. benefits. However, the numerical redn. in time to clin. improvement in those treated earlier requires confirmation in larger studies. Chinese Academy of Medical Sciences Emergency Project of COVID-19, National Key Research and Development Program of China, the Beijing Science and Technol. Project.
- 38Madhubala, R. (1997) Thin-Layer Chromatographic Method for Assaying Polyamines. Methods Mol. Biol. 79, 131– 136, DOI: 10.1385/0-89603-448-8:131There is no corresponding record for this reference.
- 39Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 9 (7), 671– 675, DOI: 10.1038/nmeth.208939NIH Image to ImageJ: 25 years of image analysisSchneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W.Nature Methods (2012), 9 (7_part1), 671-675CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the anal. of scientific images. We discuss the origins, challenges and solns. of these two programs, and how their history can serve to advise and inform other software projects.