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

2020; Elsevier BV; Volume: 295; Issue: 47 Linguagem: Inglês

10.1074/jbc.ac120.015720

ISSN

1083-351X

Autores

Egor P. Tchesnokov, Calvin J. Gordon, Emma Woolner, Dana Kocinkova, Jason K. Perry, Joy Y. Feng, Danielle Porter, Matthias Götte,

Tópico(s)

COVID-19 Clinical Research Studies

Resumo

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. 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. Correction: Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of actionJournal of Biological ChemistryVol. 297Issue 2PreviewVOLUME 295 (2020), PAGES 16156–16165 Full-Text PDF Open Access The U.S. Food and Drug Administration has recently issued an emergency use authorization (EUA) for the investigational drug remdesivir (RDV) to treat infection with severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) (1U.S. Food and Drug Administration. (2020) Fact Sheet for Health Care Providers Emergency Use Authorization (EUA) of Veklury, ® (remdesivir), pp. 1–20. Food and Drug Administration, Silver Spring, MD.Google Scholar). The EUA was largely based on a randomized clinical trial that showed a significant reduction in the time of recovery of hospitalized individuals diagnosed with coronavirus disease 2019 (COVID-19) (2Wang 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. et al.Remdesivir in adults with severe COVID-19: A randomised, double-blind, placebo-controlled, multicentre trial.Lancet. 2020; 395 (32423584): 1569-157810.1016/S0140-6736(20)31022-9Abstract Full Text Full Text PDF PubMed Scopus (2220) Google Scholar). RDV is a nucleotide analog prodrug that was designed to target the RNA-dependent RNA polymerase (RdRp) of RNA viruses (3Lo M.K. Jordan R. Arvey A. Sudhamsu J. Shrivastava-Ranjan P. Hotard A.L. Flint M. McMullan L.K. Siegel D. Clarke M.O. Mackman R.L. Hui H.C. Perron M. Ray A.S. Cihlar T. et al.GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.Sci. Rep. 2017; 7 (28262699): 4339510.1038/srep43395Crossref PubMed Scopus (311) Google Scholar, 4Siegel D. Hui H.C. Doerffler E. Clarke M.O. Chun K. Zhang L. Neville S. Carra E. Lew W. Ross B. Wang Q. Wolfe L. Jordan R. Soloveva V. Knox J. et al.Discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[2,1-f][triazin-4-amino] adenine c-nucleoside (GS-5734) for the treatment of Ebola and emerging viruses.J. Med. Chem. 2017; 60 (28124907): 1648-166110.1021/acs.jmedchem.6b01594Crossref PubMed Scopus (434) Google Scholar). The triphosphate form of RDV (RDV-TP) is an analog of the natural adenosine triphosphate (ATP) with 1′-C-nucleoside bond and a 1′-cyano-substitution. RDV has shown a broad-spectrum of antiviral activity in cell culture and animal models against negative-sense RNA viruses of the Filoviridae (e.g. Ebola virus) and Paramyxoviridae (e.g. Nipah virus and measles virus) as well as in vitro activity against viruses in the Pneumoviridae (e.g. respiratory syncytial virus) family (3Lo M.K. Jordan R. Arvey A. Sudhamsu J. Shrivastava-Ranjan P. Hotard A.L. Flint M. McMullan L.K. Siegel D. Clarke M.O. Mackman R.L. Hui H.C. Perron M. Ray A.S. Cihlar T. et al.GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.Sci. Rep. 2017; 7 (28262699): 4339510.1038/srep43395Crossref PubMed Scopus (311) Google Scholar, 4Siegel D. Hui H.C. Doerffler E. Clarke M.O. Chun K. Zhang L. Neville S. Carra E. Lew W. Ross B. Wang Q. Wolfe L. Jordan R. Soloveva V. Knox J. et al.Discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[2,1-f][triazin-4-amino] adenine c-nucleoside (GS-5734) for the treatment of Ebola and emerging viruses.J. Med. Chem. 2017; 60 (28124907): 1648-166110.1021/acs.jmedchem.6b01594Crossref PubMed Scopus (434) Google Scholar, 5Warren T.K. Jordan R. Lo M.K. Ray A.S. Mackman R.L. Soloveva V. Siegel D. Perron M. Bannister R. Hui H.C. Larson N. Strickley R. Wells J. Stuthman K.S. Van Tongeren S.A. et al.Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.Nature. 2016; 531 (26934220): 381-38510.1038/nature17180Crossref PubMed Scopus (1005) Google Scholar, 6Jordan P.C. Liu C. Raynaud P. Lo M.K. Spiropoulou C.F. Symons J.A. Beigelman L. Deval J. Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase.PLoS Pathog. 2018; 14 (29425244): e100688910.1371/journal.ppat.1006889Crossref PubMed Scopus (82) Google Scholar, 7Lo M.K. Feldmann F. Gary J.M. Jordan R. 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Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase.Antiviral Res. 2019; 169 (31233808): 10454110.1016/j.antiviral.2019.104541Crossref PubMed Scopus (321) Google Scholar, 10de Wit E. Feldmann F. Cronin J. Jordan R. Okumura A. Thomas T. Scott D. Cihlar T. Feldmann H. Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117 (32054787): 6771-677610.1073/pnas.1922083117Crossref PubMed Scopus (610) Google Scholar, 11Sheahan 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. et al.Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.Sci. Transl. Med. 2017; 9 (28659436): eaal365310.1126/scitranslmed.aal3653Crossref PubMed Scopus (1067) Google Scholar, 12Pruijssers A.J. George A.S. Schäfer A. Leist S.R. Gralinksi L.E. Dinnon 3rd, K.H. Yount B.L. Agostini M.L. Stevens L.J. Chappell J.D. Lu X. Hughes T.M. Gully K. Martinez D.R. Brown A.J. et al.Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.Cell Rep. 2020; 32 (32668216): 10794010.1016/j.celrep.2020.107940Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Several previous studies have demonstrated prophylactic and therapeutic efficacy in animal models of SARS-CoV, Middle East respiratory syndrome (MERS)-CoV and SARS–CoV-2 (10de Wit E. Feldmann F. Cronin J. Jordan R. Okumura A. Thomas T. Scott D. Cihlar T. Feldmann H. Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117 (32054787): 6771-677610.1073/pnas.1922083117Crossref PubMed Scopus (610) Google Scholar, 12Pruijssers A.J. George A.S. Schäfer A. Leist S.R. Gralinksi L.E. Dinnon 3rd, K.H. Yount B.L. Agostini M.L. Stevens L.J. Chappell J.D. Lu X. Hughes T.M. Gully K. Martinez D.R. Brown A.J. et al.Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.Cell Rep. 2020; 32 (32668216): 10794010.1016/j.celrep.2020.107940Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Low 50% effective concentrations (EC50) and a high barrier to the development of resistance were observed in cell cultures (8Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X. Smith E.C. Case J.B. Feng J.Y. Jordan R. Ray A.S. Cihlar T. Siegel D. Mackman R.L. Clarke M.O. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.mBio. 2018; 9 (29511076): e00218-e0022110.1128/mBio.00221-18Crossref Scopus (935) Google Scholar). EC50 values in the lower nanomolar range have been measured for each of these three viruses, depending on the cell type–specific metabolism of RDV-TP (12Pruijssers A.J. George A.S. Schäfer A. Leist S.R. Gralinksi L.E. Dinnon 3rd, K.H. Yount B.L. Agostini M.L. Stevens L.J. Chappell J.D. Lu X. Hughes T.M. Gully K. Martinez D.R. Brown A.J. et al.Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.Cell Rep. 2020; 32 (32668216): 10794010.1016/j.celrep.2020.107940Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In vitro selection experiments with the mouse hepatitis virus (MHV) revealed two mutations in the RdRp enzyme that confer low-level resistance to RDV (8Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X. Smith E.C. Case J.B. Feng J.Y. Jordan R. Ray A.S. Cihlar T. Siegel D. Mackman R.L. Clarke M.O. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.mBio. 2018; 9 (29511076): e00218-e0022110.1128/mBio.00221-18Crossref Scopus (935) Google Scholar). F476L and V553L were shown to cause 2.4-fold resistance and 5.0-fold resistance, respectively. The F476L+V553L double mutant showed 5.5-fold resistance to RDV. The equivalent mutations F480L and V557L introduced together in SARS-CoV were also shown to confer low-level (6.0-fold) resistance to the drug (8Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X. Smith E.C. Case J.B. Feng J.Y. Jordan R. Ray A.S. Cihlar T. Siegel D. Mackman R.L. Clarke M.O. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.mBio. 2018; 9 (29511076): e00218-e0022110.1128/mBio.00221-18Crossref Scopus (935) Google Scholar). Resistance data for SARS–CoV-2 are pending. Despite progress, there are gaps in our understanding of the mechanism of action of RDV. A key element of a refined mechanism of action is based on high rates of incorporation of RDV-TP, which translates in efficient competition with its natural counterpart ATP (13Gordon C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Götte M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.J. Biol. Chem. 2020; 295 (32094225): 4773-477910.1074/jbc.AC120.013056Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). However, the existence of a 3′-OH group allows further nucleotide incorporation events to occur and inhibition of RNA synthesis is not immediately evident. Biochemical experiments with various recombinant RdRp complexes from both minus-sense and plus-sense RNA viruses show delayed chain termination (5Warren T.K. Jordan R. Lo M.K. Ray A.S. Mackman R.L. Soloveva V. Siegel D. Perron M. Bannister R. Hui H.C. Larson N. Strickley R. Wells J. Stuthman K.S. Van Tongeren S.A. et al.Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.Nature. 2016; 531 (26934220): 381-38510.1038/nature17180Crossref PubMed Scopus (1005) Google Scholar, 6Jordan P.C. Liu C. Raynaud P. Lo M.K. Spiropoulou C.F. Symons J.A. Beigelman L. Deval J. Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase.PLoS Pathog. 2018; 14 (29425244): e100688910.1371/journal.ppat.1006889Crossref PubMed Scopus (82) Google Scholar, 13Gordon C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Götte M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.J. Biol. Chem. 2020; 295 (32094225): 4773-477910.1074/jbc.AC120.013056Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). RdRp complexes of SARS-CoV, MERS-CoV, and SARS–CoV-2 stop RNA synthesis after position i + 3, i.e. three nucleotides following incorporation of RDV-TP at position i (13Gordon C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Götte M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.J. Biol. Chem. 2020; 295 (32094225): 4773-477910.1074/jbc.AC120.013056Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). This pattern is identical with the three RdRp complexes, which points to a common mechanism of inhibition. Modeling studies suggested that a steric clash between the side chain of the conserved Ser-861 and the 1′-CN group of the incorporated RDV prevent translocation, which means that the nucleotide binding site at position i + 4 is still occupied with the 3′-end of the primer (14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Another important observation is that RNA synthesis arrest can be overcome with higher concentrations of natural nucleotide pools (14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Increasing the NTP concentrations gradually reduces termination efficiency and favors read-through to yield the full-length RNA product. Efficient read-through is seen at concentrations as low as 10 μm. Intracellular NTP concentrations are in the high μm and low mm range (15Traut T.W. Physiological concentrations of purines and pyrimidines.Mol. Cell. Biochem. 1994; 140 (7877593): 1-2210.1007/BF00928361Crossref PubMed Scopus (1302) Google Scholar, 16Kennedy E.M. Gavegnano C. Nguyen L. Slater R. Lucas A. Fromentin E. Schinazi R.F. Kim B. Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages.J. Biol. Chem. 2010; 285 (20924117): 39380-3939110.1074/jbc.M110.178582Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), which suggests that read-through reactions likely occur under biologically relevant conditions. It is therefore conceivable that the primer strand, and by extension the entire negative-sense copy of the genome, contains several RDV residues. Although the CoV exonuclease (ExoN) exhibits 3′–5′ proofreading activity (8Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X. Smith E.C. Case J.B. Feng J.Y. Jordan R. Ray A.S. Cihlar T. Siegel D. Mackman R.L. Clarke M.O. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.mBio. 2018; 9 (29511076): e00218-e0022110.1128/mBio.00221-18Crossref Scopus (935) Google Scholar, 17Minskaia E. Hertzig T. Gorbalenya A.E. Campanacci V. Cambillau C. Canard B. Ziebuhr J. 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This scenario raises the question whether RDV residues embedded in the template strand may also cause inhibition during synthesis of the second RNA strand, i.e. during transcription or viral genome synthesis. To address this question, we have devised a method that allows synthesis of small RNA model templates with RDV-TP incorporated at a single strategic position. RNA synthesis with recombinant SARS–CoV-2 RdRp shows that incorporation of UTP opposite RDV and the following nucleotide incorporation are compromised. Similar to earlier observations with the primer strand, this inhibition can be overcome with increasing NTP concentrations, although the threshold is much higher here. This effect is partially reversed with a known resistance-conferring mutation that can affect the positioning of the template strand. Based on these data, we propose a comprehensive mechanism of action of RDV that involves both the primer strand and the template. Several recent structural and biochemical studies have demonstrated that the active SARS–CoV-2 RdRp complex is composed of the three nonstructural proteins nsp7, nsp8, and nsp12 (20Subissi L. Posthuma C.C. Collet A. Zevenhoven-Dobbe J.C. Gorbalenya A.E. Decroly E. Snijder E.J. Canard B. Imbert I. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25197083): E3900-E390910.1073/pnas.1323705111Crossref PubMed Scopus (352) Google Scholar, 21Kirchdoerfer R.N. Ward A.B. Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.Nat. Commun. 2019; 10 (31138817): 234210.1038/s41467-019-10280-3Crossref PubMed Scopus (501) Google Scholar, 22Hillen H.S. Kokic G. Farnung L. Dienemann C. Tegunov D. Cramer P. 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We utilized constructs that co-express the viral protease nsp5 together with nsp7, nsp8, and nsp12 in insect cells (13Gordon C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Götte M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.J. Biol. Chem. 2020; 295 (32094225): 4773-477910.1074/jbc.AC120.013056Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Active complexes were captured via the histidine-tagged nsp8, and binary complexes composed of nsp8 and nsp12 were identified with Coomassie Blue staining. Here we show that overloading of the gel with the soluble protein sample also visualizes nsp7 (Fig. S1). Using the same construct design, we generated complexes with mutations at position Ser-861 in nsp12 that are expected to affect efficiency of delayed chain termination with RDV-TP. The steric clash hypothesis predicts that smaller side chains will reduce delayed chain termination and larger side chains will increase inhibition, provided that substitutions do not impede regular NTP incorporations. We compared RNA synthesis of the WT SARS–CoV-2 RdRp complex with S861G, S861A, and S861P mutants (Fig. 1). Reactions were monitored on a 17-mer RNA template that contains a single site of incorporation for RDV-TP at position 9 or i (Fig. 1A). The WT enzyme shows delayed chain termination at position 12 (i + 3) (Fig. 1A), which is illustrated by more than 95% reduction in the full template length-to–(i + 3) product ratio in the presence of RDV-TP as compared with the full template length-to–p12 product ratio in the presence of ATP (Fig. 1B). Termination efficiency is gradually reduced by increasing the concentration of the next incoming nucleotide (UTP), as previously reported (14Gordon C.J. Tchesnokov E.P. Woolner E. Perry J.K. Feng J.Y. Porter D.P. Götte M. Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.J. Biol. Chem. 2020; 295 (32284326): 6785-679710.1074/jbc.RA120.013679Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). 50% read-through is seen at UTP concentrations of ∼0.3 μm, 90% read-through is seen with ∼3 μm, and inhibition is abolished with ∼30 μm (Fig. 1C). Delayed chain termination is not observed with the S861G mutant. These findings are consistent with the removal of a steric clash when serine is replaced with glycine. Full-length product formation is already seen to near completion at UTP concentrations of ∼1 μm (Fig. 1A). The S861A mutation shows only subtle reductions in delayed chain termination (Fig. 1, A and B), which is in agreement with a previous report by Wang and colleagues (23Wang Q. Wu J. Wang H. Gao Y. Liu Q. Mu A. Ji W. Yan L. Zhu Y. Zhu C. Fang X. Yang X. Huang Y. Gao H. Liu F. et al.Structural basis for RNA replication by the SARS-CoV-2 polymerase.Cell. 2020; 182 (32526208): 417-428e41310.1016/j.cell.2020.05.034Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). We further show that the bulkier side chain of S861P shows subtle increases in UTP concentrations required to overcome delayed chain termination (Fig. 1, A and C). The high efficiency of read-through with increasing NTP concentrations suggests that the newly synthesized copy of the RNA genome contains several RDV residues. This raises the question whether RDV may also inhibit RNA synthesis when present in the template (Fig. 2). The challenge to synthesize sufficient amounts of RNAs with embedded RDV residues using RdRp is the need for strand separation. Hence, we developed a protocol to generate single-stranded RNAs using T7 RNA polymerase along a DNA template that can be selectively degraded (see "Experimental procedures"). We synthesized two 20-mer model RNAs that contain either adenosine (Template A) or RDV (Template R) at the same strategic position 11 (Fig. S2). A 4-mer primer was used to initiate RNA synthesis with SARS–CoV-2 RdRp in the presence of [α-32P]GTP (Fig. 2A). With Template A, a mixture of minimal concentrations of 0.1 μm CTP and UTP, and 1 μm ATP yields predominantly the full-length 20-mer RNA product (Fig. 2B). Increasing the concentration of UTP shows no significant increases in RNA synthesis. With Template R, we observe an intermediate product at position 10 at the same base concentrations of CTP, UTP, and ATP (Fig. 2B). These findings demonstrate that 0.1 μm UTP is not sufficient for incorporation opposite RDV, which is a marked difference to results obtained with Template A. Increasing concentrations of UTP can overcome the inhibitory effects, and we observe an accumulation of product at position 11, which points to a second site of inhibition immediately after the template-embedded RDV. A similar effect is seen with increasing the concentration of UTP and CTP that is required later at position 14. Increases only in CTP did not yield significant amounts of product at position 11, suggesting that incorporation of CTP opposite RDV is negligible. Higher concentrations of UTP and ATP or UTP, ATP, and CTP gradually reduced the inhibitory effects and increased the yield of full-length product. Overall inhibition is driven by the accumulation of products at positions 10 and 11. Together these findings show that a single RDV residue in the template inhibits efficiency of incorporation of the complementary UTP and the adjacent NTP (ATP in this particular sequence context). NTP concentrations required to overcome these obstacles are higher than observed with delayed chain termination (Fig. 2C). We next asked whether such template-dependent inhibition provides a biologically relevant mechanism of action of RDV. To address this question we attempted to study possible neutralizing effects of known resistance conferring mutations. In nsp12, two amino acid substitutions have been associated with low-level resistance to RDV (8Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X. Smith E.C. Case J.B. Feng J.Y. Jordan R. Ray A.S. Cihlar T. Siegel D. Mackman R.L. Clarke M.O. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofrea

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