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 Ser ⁷⁵⁹ -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.

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.

Yellow fever virus (YFV) is a deadly mosquito-borne flavivirus that infects approximately 200,000 individuals each year. YFV outbreak and infection are persistent throughout South America and Africa, demonstrating significant epidemic potential. Although an approved and effective vaccine exists, the zoonotic nature of YFV undermines any potential eradication efforts, highlighting the need for effective, direct-acting antivirals. Essential for viral replication, RNA-dependent RNA polymerase (RdRp) is a proven therapeutic target. A prominent example includes sofosbuvir, a 2'-α-fluoro,2'-β-methyluridine prodrug approved for the treatment of hepatitis C virus (HCV), that has demonstrated efficacy against YFV in vitro. A structurally similar 2'-α-fluoro, 2'-β-bromouridine prodrug has exhibited potent anti-YFV activity both in vitro and in vivo. Here, we expressed the full-length nonstructural protein 5 from YFV in insect cells to investigate the active triphosphate form of these prodrugs. Enzyme kinetics indicate that both nucleotide analogs are incorporated less efficiently than UTP. Once incorporated, the analogs inhibit RNA synthesis through immediate chain termination. Omitting the 2'-β-modification alleviates the inhibition of RNA synthesis, highlighting its role in eliciting an antiviral effect. S282T is a well-characterized mutation in motif B of HCV RdRp that confers resistance to sofosbuvir. We discovered that the analogous substitution in YFV (S603T) improved discrimination against the 2'-α-fluoro,2'-β-modified uridine analogs. Collectively, our findings explain their observed anti-YFV activity and identify a conserved mechanism of resistance. Based on its in vivo efficacy and mechanism of action, the 2'-fluoro,2'-bromouridine prodrug shows potential for future therapeutic strategies against YFV.

Nucleoside analogs are successful in treating viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when embedded in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM structures demonstrate that SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) preferentially incorporates remdesivir triphosphate (RTP), outcompeting 10-fold excess ATP; however, successive RTP incorporations are disfavored when ATP is present. The RdRp structures demonstrate that 1′-cyano-imposed conformational restriction of the remdesivir:UMP base-pair is resistant to translocation, reducing successive RTP incorporations. The S759A mutant confers RTP resistance. We show that the mutation switches the RdRp preference to ATP; RTP is incorporated only at 10-fold excess to ATP. The structures of S759A RdRp reveal that the primer 3′-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. These findings have implications for designing non-obligate nucleoside analogs with different inhibition mechanisms.

Flaviviruses are arthropod-borne RNA viruses associated with significant human diseases globally. There are no effective direct-acting antivirals approved to treat these viral infections. Given its critical role in viral replication, the RNA-dependent RNA polymerase (RdRp) is a logical target for antiviral drug development. Remdesivir (formerly GS-5734), a 1′-cyano modified C-adenosine monophosphate prodrug, was the first US Food and Drug Administration (FDA) approved antiviral for coronavirus disease 2019 (COVID-19) and was also shown to inhibit flavivirus replication. GS-7682, a 4′-cyano modified C-adenosine prodrug, exhibits a broad-spectrum antiviral activity. Here, we determined the anti-flavivirus potency of both remdesivir and GS-7682 and characterized their active triphosphate forms, GS-443902 and GS-646939, respectively, against a panel of purified flavivirus RdRps. These include dengue, Japanese encephalitis, West Nile, yellow fever, and Zika. Enzyme kinetics demonstrate efficient RNA incorporation of GS-443902 and GS-646939. GS-646939 acts as an immediate chain terminator. Conversely, GS-443902 acts through a template-dependent inhibition mechanism by impeding the incorporation of the complementary UTP. Both mechanisms correlate with anti-flavivirus activity, although remdesivir is generally superior. The data demonstrate that immediate chain termination is not necessarily a preferred mechanism of action of nucleotide analogs. Template-dependent inhibition should also be considered, especially for viruses lacking intrinsic proofreading activities.