No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002-2003, and the recent emergence of ...Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN-) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN- CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN- population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition, and suggest that small-molecule inhibitors of ExoN activity could be potential pan-CoV therapeutics in combination with RBV or RNA mutagens.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the ...mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease associated with more than 2468 human infections and over 851 deaths in 27 countries ...since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathology. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathology. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.
Much beyond this rate, RNA viruses risk crossing an "error threshold," or the point at which there are too many deleterious mutations for the viral population to reproduce faithfully 4. ...while ...allowing for enormous population diversity, the low-fidelity of RdRp-mediated replication imposes constraints on both viral genome size and maintenance of genomic integrity, theoretically limiting the size of RNA virus genomes to around ~15 kb (reviewed in 4). ...the probability that fidelity is regulated by a multi-protein complex involving nsp12-RdRp, nsp14-ExoN, and nsp10 suggests that interference with fidelity could be achieved through the disruption of multiple enzymatic activities and/or protein-protein interactions.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral ...therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC
values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.
•Coronaviruses (CoV) represent current and future risk for pandemic zoonotic infections.•Nucleoside analogues are highly active across multiple virus families.•CoVs encode a proofreading ...exoribonuclease that opposes inhibition by many nucleoside analogues.•Remdesivir is an adenosine analogue that acts against a broad spectrum of CoVs.•Other nucleoside analogues show promise against CoVs.
Recent outbreaks of SARS-Coronavirus and MERS-Coronavirus (CoV) have heightened awareness about the lack of vaccines or antiviral compounds approved for prevention or treatment of human or potential zoonotic CoVs. Anti-CoV drug development has long been challenged by the activity of a 3′ to 5′ proofreading exoribonuclease unique to CoVs. Recently, a promising nucleoside analogue with broad-spectrum activity against CoVs has been identified. This review will discuss progress made in the development of antiviral nucleoside and nucleotide analogues targeting viral RNA synthesis as effective therapeutics against CoV infections and propose promising strategies for combination therapy.
Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As ...in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.
Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. ...However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due ...to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs
and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC
) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC
The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same
resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.
Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV
and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.
The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no ...antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-
d
-
N
4
-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.
Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3′-5′ exoribonuclease (ExoN). β-
d
-
N
4
-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration EC
50
= 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC
50
= 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.
IMPORTANCE
The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-
d
-
N
4
-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.
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