Structural and Functional Basis of the Fidelity of Nucleotide Selection by Flavivirus RNA-Dependent RNA Polymerases

Selisko, Barbara; Papageorgiou, Nicolas; Ferrón, François; Canard, Bruno

doi:10.3390/v10020059

Cited by 53 publications

(57 citation statements)

References 95 publications

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“…This polymerase displays similar catalytic mechanisms and some key conserved amino acids in the active site among different positive sense RNA viruses, to which coronaviruses and HCV belong. 4 Like RdRps in other viruses, the coronavirus enzyme is highly error-prone, 5 which might increase its ability to accept modified nucleotide analogues as substrates. Nucleotide and nucleoside analogues that inhibit polymerases are an important group of anti-viral agents.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Chien

Anderson

Jockusch

et al. 2020

Preprint

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SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). Here, using polymerase extension experiments, we have demonstrated that the active triphosphate form of Sofosbuvir (a key component of the FDA approved hepatitis C drug EPCLUSA), is incorporated by SARS-CoV-2 RdRp, and blocks further incorporation. Using the same molecular insight, we selected the active triphosphate forms of three other anti-viral agents, Alovudine, AZT (an FDA approved HIV/AIDS drug) and Tenofovir alafenamide (TAF, an FDA approved drug for HIV and hepatitis B) for evaluation as inhibitors of SARS-CoV-2 RdRp. We demonstrated the ability of these three viral polymerase inhibitors, 3'-fluoro-3'-deoxythymidine triphosphate, 3'-azido-3'deoxythymidine triphosphate and Tenofovir diphosphate (the active triphosphate forms of Alovudine, AZT and TAF, respectively) to be incorporated by SARS-CoV-2 RdRp, where they also terminate further polymerase extension. These results offer a strong molecular basis for these nucleotide analogues to be evaluated as potential therapeutics for COVID-19.

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Section: Introductionmentioning

confidence: 99%

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Chien

Anderson

Jockusch

et al. 2020

Preprint

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“…RdRp in coronaviruses is a precise and welldefined drug target; the active site of the RdRp is highly conserved among different positive-sense RNA viruses and shares common structural features in these viruses. 4 RdRps, including the coronavirus polymerase, have low fidelity, 5 which enables them to recognize a variety of modified nucleotide analogues as substrates. Such nucleotide and nucleoside analogues may inhibit further RNA-polymerase catalyzed RNA replication and are therefore important candidate anti-viral agents.…”

Section: Introductionmentioning

confidence: 99%

Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase

Jockusch

Tao

et al. 2020

Preprint

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SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. We previously demonstrated that four nucleotide analogues (specifically, the active triphosphate forms of Sofosbuvir, Alovudine, AZT and Tenofovir alafenamide) inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Tenofovir and emtricitabine are the two components in DESCOVY and TRUVADA, the two FDA-approved medications for use as pre-exposure prophylaxis (PrEP) to prevent HIV infection. This is a preventative method in which individuals who are HIV negative (but at high-risk of contracting the virus) take the combination drug daily to reduce the chance of becoming infected with HIV. PrEP can stop HIV from replicating and spreading throughout the body. We report here that the triphosphates of tenofovir and emtricitabine, the two components in DESCOVY and TRUVADA, act as terminators for the SARS-CoV-2 RdRp catalyzed reaction. These results provide a molecular basis to evaluate the potential of DESCOVY and TRUVADA as PrEP for COVID-19.

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“…Each Motif V mutation was designed to interrupt specific WT residue-residue interactions or residue-substrate interactions. As a negative control, D664 in the NS5 polymerase catalytic active site was mutated to valine (D664V) in order to disrupt viral genome replication (42)(43)(44)(45). Additionally, the NS3 mutants A286L (Motif II) and R387M (Motif IVa) were used as controls for ablating ATPase activity and RNA binding affinity, respectively (30,(46)(47)(48)(49)(50).…”

Section: Motif V Mutants Affect Viral Genome Replication -As Previousmentioning

confidence: 99%

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

Davidson

McCullagh

et al. 2019

Preprint

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The unwinding of double-stranded RNA intermediates is a critical component for the replication of flavivirus RNA genomes. This function is achieved by the C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP and RNA binding pockets is not well understood. Previous molecular dynamics simulations published by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. In order to investigate the role of Motif V in this process, a study combining molecular dynamics, biochemistry and virology has been employed.Mutations of Motif V were tested in both replicon and recombinant protein systems to investigate viral genome replication, RNA binding affinity, ATP hydrolysis activity and helicase unwinding activity. Using these analyses, we found that T407A and S411A in Motif V demonstrated increased turnover rates, suggesting that the mutations causes the helicase to unwind dsRNA more quickly than WT. Additionally, simulations of each mutant were used to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP binding pocket and the helical gate. These data help define the linkage between ATP hydrolysis and helicase activity within NS3 and provide insight into the biophysical mechanisms for ATPase driven NS3 helicase function. Arthropod-borne flaviviruses, such as yellow fever virus, Japanese encephalitis virus, Zika virus, WestNile virus (WNV) and dengue virus, are a major health concern in the tropical and subtropical regions of the world (1, 2). Infection from these viruses cause disease symptoms ranging from flu-like illness to encephalitis, hemorrhagic fever, coma, and potentially death (3). Over half of the world population is at risk for infection from one or more of these viruses (4, 5). Dengue virus, specifically, infects around 50 million people each year, and of those individuals, 20,000 contract dengue hemorrhagic fever leading to their mortality (6). Additionally, WNV over the past 20 years within the 48 continental United States has around 50,000 clinical infections with a 5% mortality rate (7-9). Currently, there are no approved antiviral drugs for treating flaviviral infections and the vaccines in circulation, like the yellow fever vaccine, are not readily available worldwide for most flaviviruses (10-12). In order to develop new antiviral treatments (drugs and vaccines) for these viruses, a fundamental understanding of how these flaviviruses replicate is required.

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Structural and Functional Basis of the Fidelity of Nucleotide Selection by Flavivirus RNA-Dependent RNA Polymerases

Cited by 53 publications

References 95 publications

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase

Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

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