scholarly journals Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Egor P. Tchesnokov ◽  
Parisa Raeisimakiani ◽  
Marianne Ngure ◽  
David Marchant ◽  
Matthias Götte
Keyword(s):  
Rna Polymerase ◽  
Ebola Virus ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex

2020 ◽  
Vol 6 (10) ◽  
pp. 2800-2811
Author(s):  
Anand Balakrishnan ◽  
Edmund Price ◽  
Catherine Luu ◽  
Jacob Shaul ◽  
Charles Wartchow ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Biochemical Characterization ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Syncytial Virus

scholarly journals Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

2020 ◽  
Vol 295 (20) ◽  
pp. 6785-6797 ◽  
Author(s):  
Calvin J. Gordon ◽  
Egor P. Tchesnokov ◽  
Emma Woolner ◽  
Jason K. Perry ◽  
Joy Y. Feng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Lassa Virus ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Direct Acting Antiviral ◽  
Nucleotide Analogue ◽  
Direct Acting

Effective treatments for coronavirus disease 2019 (COVID-19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERS-CoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2′-C-methylated compounds, including sofosbuvir, approved for the management of hepatitis C virus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination of RNA synthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral.

scholarly journals Nonnucleoside Inhibitor of Measles Virus RNA-Dependent RNA Polymerase Complex Activity

2007 ◽  
Vol 51 (7) ◽  
pp. 2293-2303 ◽  
Author(s):  
Laura K. White ◽  
Jeong-Joong Yoon ◽  
Jin K. Lee ◽  
Aiming Sun ◽  
Yuhong Du ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput Screening ◽  
Measles Virus ◽  
Specific Inhibitor ◽  
Cross Resistance ◽  
Complex Activity ◽  
Rna Dependent Rna Polymerase ◽  
Developmental Potential ◽  
Polymerase Complex ◽  
Fusion Inhibitors

ABSTRACT Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex.

scholarly journals Potent Non-Nucleoside Inhibitors of the Measles Virus RNA-Dependent RNA Polymerase Complex

2008 ◽  
Vol 51 (13) ◽  
pp. 3731-3741 ◽  
Author(s):  
Aiming Sun ◽  
Jeong-Joong Yoon ◽  
Yan Yin ◽  
Andrew Prussia ◽  
Yutao Yang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Measles Virus ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Nucleoside Inhibitors

scholarly journals Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

2020 ◽  
Author(s):  
Jack PK Bravo ◽  
Tyler L Dangerfield ◽  
David W Taylor ◽  
Kenneth A Johnson
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rational Design ◽  
Antiviral Drugs ◽  
Chain Termination ◽  
Nucleoside Analog ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

scholarly journals Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 326 ◽  
Author(s):  
Egor Tchesnokov ◽  
Joy Feng ◽  
Danielle Porter ◽  
Matthias Götte
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Virus Disease ◽  
Chain Termination ◽  
Mitochondrial Rna ◽  
Long Distance ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogue ◽  
Nucleotide Incorporation

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

scholarly journals Naturally Occurring Single Mutations in Ebola Virus Observably Impact Infectivity

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Gary Wong ◽  
Shihua He ◽  
Anders Leung ◽  
Wenguang Cao ◽  
Yuhai Bi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rational Design ◽  
Ebola Virus ◽  
Viral Fitness ◽  
Rna Dependent Rna Polymerase ◽  
Time To Death ◽  
Recombinant Viruses ◽  
Antiviral Therapies ◽  
Naturally Occurring ◽  
Huh7 Cells

ABSTRACT Sequencing of Ebola virus (EBOV) genomes during the 2014–2016 epidemic identified several naturally occurring, dominant mutations potentially impacting virulence or tropism. In this study, we characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA polymerase (L). Compared with the wild-type (WT) EBOV C07 isolate, NP and L mutants conferred a replication advantage in monkey Vero E6, human A549, and insectivorous bat Tb1.Lu cells, while L mutants displayed a disadvantage in human Huh7 cells. The replication of the GP mutant was significantly delayed in Tb1.Lu cells and similar to that of the WT in other cells. The L mutant was less virulent, as evidenced by increased survival for mice and a significantly delayed time to death for ferrets, but increased lengths of the period of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework for the study of other mutations. IMPORTANCE During the Ebola virus (EBOV) disease outbreak in West Africa in 2014–2016, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein [GP], nucleoprotein [NP], or RNA-dependent RNA polymerase [L]) in cell culture, as well as in mice and ferrets, by generating recombinant viruses (based on an early West African EBOV strain) each carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas the GP mutation slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.

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