The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus

Abstract

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.

Keywords: Ebola virus (EBOV); Middle East respiratory syndrome coronavirus (MERS–CoV); RNA chain termination; RNA-dependent RNA polymerase (RdRp); SARS–CoV-2; antiviral drug; coronavirus; drug development; enzyme inhibitor; nucleoside/nucleotide analog; plus-stranded RNA virus; positive-sense RNA virus; remdesivir; viral polymerase; viral replicase.

Figure 1.

Expression, purification, and characterization of the MERS RdRp complex. A, the construct contains nonstructural proteins nsp5, nsp7, nsp8, and nsp12. Red rectangles indicate original nsp5 protease cleavage sites. His₈ and Strep indicate the locations of histidine and strep tags, respectively. B, a snapshot of a sequence alignment (T-Coffee) of representative RdRp enzymes from positive-sense RNA genome viruses illustrating sequence conservation within RdRp motif C. C, SDS-PAGE migration pattern of the purified enzyme preparations stained with Coomassie Brilliant Blue G-250 dye. Proteins migrating at ∼100 and ∼25 kDa contain nsp12 and nsp8, respectively. D, RNA synthesis on a short model primer/template substrate. Template and primer were both phosphorylated (p) at their 5′-ends. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. RNA synthesis was monitored with the purified MERS RdRp complex wt (motif C = SDD) and active-site mutant (motif C = SNN) in the presence of NTP combinations designed to generate specific products. Lanes m illustrate the migration pattern of the radiolabeled 4-nucleotide-long primer. HCV, hepatitis C virus; NoV, norovirus; PoV, poliovirus.

Figure 2.

Patterns of inhibition of RNA synthesis with RDV-TP. A, RNA primer/template substrates used to test multiple (left panel) or single (right panel) incorporations of RDV-TP. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. i indicates incorporation site for the first (left panel) or the only (right panel) RDV-TP. Full length indicates the full-template length products of RNA synthesis. B, RDV-TP incorporation was monitored with purified EBOV and MERS RdRp complexes in the presence of the indicated combinations of NTPs and RDV-TP.

Figure 3.

Competition between RDV-TP and ATP. A, the RNA primer/template substrate used in this assay is shown above the gel. 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 MERS RdRp complex in the presence of 0.02 μm ATP, CTP, and UTP mix and increasing concentrations of RDV-TP as indicated. B, graphic representation and IC₅₀ determination fitting of quantified data from A. The error bars represent standard deviation of the data within four independent experiments. C, graphic representation of the relationship between IC₅₀ values for RDV-TP measured at different NTP concentrations. The average IC₅₀ values for RDV-TP are shown above the corresponding bars. The error bars represent standard deviation of the data within at least three independent experiments.

Figure 4.

Chemical structures of ATP and ATP analogs.