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 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 Remdesivir and Ledipasvir among the FDA-Approved Antiviral Drugs Have Potential to Inhibit SARS-CoV-2 Replication

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1052
Author(s):  
Rameez Hassan Pirzada ◽  
Muhammad Haseeb ◽  
Maria Batool ◽  
MoonSuk Kim ◽  
Sangdun Choi
Keyword(s):  
Antiviral Drugs ◽  
Effective Concentration ◽  
Nonstructural Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Direct Acting Antiviral ◽  
Rapid Spread ◽  
Half Maximal Effective Concentration ◽  
Dynamics Simulations ◽  
Direct Acting ◽  
Transcription And Replication

The rapid spread of the virus, the surge in the number of deaths, and the unavailability of specific SARS-CoV-2 drugs thus far necessitate the identification of drugs with anti-COVID-19 activity. SARS-CoV-2 enters the host cell and assembles a multisubunit RNA-dependent RNA polymerase (RdRp) complex of viral nonstructural proteins that plays a substantial role in the transcription and replication of the viral genome. Therefore, RdRp is among the most suitable targets in RNA viruses. Our aim was to investigate the FDA approved antiviral drugs having potential to inhibit the viral replication. The methodology adopted was virtual screening and docking of FDA-approved antiviral drugs into the RdRp protein. Top hits were selected and subjected to molecular dynamics simulations to understand the dynamics of RdRp in complex with these drugs. The antiviral activity of the drugs against SARS-CoV-2 was assessed in Vero E6 cells. Notably, both remdesivir (half-maximal effective concentration (EC50) 6.6 μM, 50% cytotoxicity concentration (CC50) > 100 µM, selectivity index (SI) = 15) and ledipasvir (EC50 34.6 μM, CC50 > 100 µM, SI > 2.9) exerted antiviral action. This study highlights the use of direct-acting antiviral drugs, alone or in combination, for better treatments of COVID-19.

scholarly journals Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action

2020 ◽  
Vol 295 (47) ◽  
pp. 16156-16165 ◽  
Author(s):  
Egor P. Tchesnokov ◽  
Calvin J. Gordon ◽  
Emma Woolner ◽  
Dana Kocinkova ◽  
Jason K. Perry ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Structural Model ◽  
Antiviral Agent ◽  
Product Formation ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Biologically Relevant ◽  
Direct Acting Antiviral ◽  
Dependent Inhibition ◽  
Direct Acting

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain termination at position i + 3. Here we demonstrate that the S861G mutation in RdRp eliminates chain termination, which confirms the existence of a steric clash between Ser-861 and the incorporated RDV-TP. With WT RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned because of a significant clash with Ala-558. The adjacent Val-557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS–CoV-2 RdRp by RDV is biologically relevant.

scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

scholarly journals Initiation of (—)-Strand RNA Synthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis of Oligonucleotides

Virology ◽  
1997 ◽  
Vol 228 (1) ◽  
pp. 121
Author(s):  
Jin-Hua Sun ◽  
Scott Adkins ◽  
Greta Faurote ◽  
C.Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Dependent Rna Polymerase

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

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