Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Mechanism and spectrum of inhibition of a 4'-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

BACKGROUND: Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. METHODS: Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. RESULTS: Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). CONCLUSIONS: The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir.

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15.

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance.

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

The Nucleoside/Nucleotide Analogs Tenofovir and Emtricitabine Are Inactive against SARS-CoV-2.

The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19.

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

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

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.

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®.

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1-6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1-6 HCV patients who have previously failed another treatment regimen.

Discovery of potent and selective inhibitors of calmodulin-dependent kinase II (CaMKII).

We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/ß). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat F = 73%) and IV pharmacological studies.

Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus.

Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.

No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Discovery of GS-7682, a Novel 4'-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex.

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

Mutations in the SARS-CoV-2 RNA-dependent RNA polymerase confer resistance to remdesivir by distinct mechanisms.

The nucleoside analog remdesivir (RDV) is a Food and Drug Administration-approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration from 2.7- to 10.4-fold. Sequence analysis identified nonsynonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I, and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first and then combined with S759A. Introduction of S759A and V792I substitutions at homologous nsp12 positions in murine hepatitis virus demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas nsp12-V792I diminished the uridine triphosphate concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro-selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.