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

Abstract

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.

Keywords: COVID-19; Ebola virus; Lassa virus; MERS; RNA polymerase; RNA-dependent RNA polymerase (RdRp); SARS; SARS-CoV-2; coronavirus (CoV); drug action; drug development; drug discovery; favipiravir; plus-stranded RNA virus; remdesivir; replication; ribavirin; sofosbuvir.

Figure 1.

Expression, purification, and characterization of the SARS-CoV and SARS-CoV-2 RdRp complexes. A, SDS-PAGE migration pattern of the purified enzyme preparations stained with Coomassie Brilliant Blue G-250 dye. Bands migrating at ∼100 kDa and ∼25 kDa contain nsp12 and nsp8, respectively. B, RNA synthesis on a short model primer/template substrate. Template and primer were both phosphorylated (p) at their 5′-ends. A radiolabeled 4-mer primer serves as a marker (m). G indicates incorporation of the radiolabeled nucleotide opposite template position 5. RNA synthesis was monitored with the purified RdRp complexes representing WT (wt, motif C = SDD) and the active-site mutant (motif C = SNN).

Figure 2.

Chemical structures of ATP and ATP, CTP, and UTP nucleotide analogues used in this study.

Figure 3.

A, X-ray structure of HCV RdRp with an incoming nonhydrolyzable ADP substrate (PDB entry 4WTD). The 2′-OH of the substrate is recognized by the trio of residues, Asp-225, Ser-282, and Asn-291, with hydrogen bonds formed to Ser-282 and Asn-291 in this preincorporation state. B, model of SARS-CoV-2 nsp12 with incoming ATP. In addition to the analogous Asp/Ser/Asn residues, Thr-680 is positioned to alter the hydrogen-bonding network and effectively pull the substrate lower into the pocket relative to NS5B. C, model of SARS-CoV-2 with SOF-TP. The greater occlusion of the 2′ position due to Asp-623 and Ser-682 makes 2′β-methyl substitution less effective than with NS5B. D, model of SARS-CoV-2 with remdesivir-TP. The remdesivir 1′CN sits in a pocket formed by residues Thr-687 and Ala-688. Residues Asp-623 and Ser-682 (not shown) adopt the same conformations as with ATP.

Figure 4.

Patterns of inhibition of RNA synthesis with RDV-TP. A, RNA primer/template substrates used to test multiple (left) or single (right) incorporations of RDV-TP. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. RDV-TP incorporation is indicated by i. RDV-TP incorporation was monitored with purified CoV RdRp complexes (B) and LASV L protein (C) in the presence of the indicated combinations of NTPs and RDV-TP.

Figure 5.

A steric clash between the incorporated RDV and Ser-861 prevents enzyme translocation at i+3. A–D, the primer with the incorporated RDV (green) translocates without obstruction from the substrate position i through i+3, allowing incorporation of three subsequent nucleotides (yellow). E, at i+4, the 1′-CN moiety of RDV encounters a steric clash with Ser-861 of nsp12. F, this clash likely prevents the enzyme from advancing into i+4.

Figure 6.

Overcoming of delayed chain termination. The RNA primer/template substrate used in this assay is shown above the gels. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of ATP or RDV-TP. RNA synthesis was monitored with purified SARS-CoV-2 RdRp complex in the presence of indicated concentrations of NTP mixtures. A, time dependence of delayed chain termination. B, overcoming delayed chain termination with increasing concentrations of UTP.

Figure 7.

Mechanism of inhibition of CoV RdRp by RDV-TP. 1, the priming strand is shown with green circles, colorless circles represent residues of the template, and the blue oval represents the active CoV RdRp complex. This is a schematic representation of a random elongation complex. The footprint of RdRp on its primer/template is unknown. 2, competition of RDV-TP with its natural counterpart ATP opposite template uridine (U). The incorporated nucleotide analogue is illustrated by the red circle. 3, RNA synthesis is terminated after the addition of three more nucleotides, which is referred to as delayed chain termination. 4, delayed chain termination can be overcome by high ratios of NTP/RDV-TP.