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.
Highlights
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)
The emergence of a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2, formerly 2019-nCoV),3 initiated a global effort to identify effective treatments focusing on agents with demonstrated antiviral activity against SARS–CoV, Middle East respiratory syndrome (MERS– CoV), or related positive-sense RNA viruses
Broad spectrum antivirals or compounds with demonstrated activity against SARS–CoV or MERS–CoV are considered for the treatment of infection caused by the novel coronavirus SARS–CoV-2
Summary
The baculovirus expression system has recently been used to produce recombinant nsp from SARS–CoV (13). These sequences were used to compare the inhibitory effects of RDV on EBOV RdRp and MERS RdRp. For EBOV RdRp, we observed delayed chain termination at position i ϩ 5 as previously described (Fig. 2B) (8). The inhibition patterns with RDV-TP differ markedly from our results with EBOV RdRp. RNA synthesis is arrested at positions i ϩ 3 and i ϩ 4 with a template that provides multiple sites of incorporation of the inhibitor, and the full-length product is only seen as a faint band. The template that allows only a single incorporation event yields RNA synthesis arrest at position i ϩ 3 and an increased amount of the full-length product. Sponding increase in IC50 values for RDV-TP (Fig. 3C), which shows that RDV-TP is a competitive inhibitor
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