Investigating Inhibition of the SARS-CoV-2 Helicase by Nucleoside Analog Antivirals and Potential Resistance Mechanisms

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Replication and transcription of the SARS-CoV-2 genome is mediated by 16 highly conserved non-structural proteins (nsp), making them prime targets for antiviral drug development. Prior studies have demonstrated that the ability of the viral RNA polymerase (nsp12) to use nucleotide triphosphates (NTP) for energy is inhibited by nucleoside analogs, compounds similar to NTPs. Subsequent research has also shown that mutations within nsp12’s site of NTP catabolism can prevent binding or incorporation of these drugs, resulting in antiviral resistance. However, little research exists studying the effects of nucleoside analogs on the viral helicase (nsp13) and whether this enzyme could exhibit the same resistance. In this paper, analysis of nsp13’s ability to unwind a double-stranded DNA molecule in vitro in the presence of nucleoside analogs shows that these drugs also inhibit the SARS-CoV-2 helicase. Since this inhibition is most effective when the nucleoside analog drug is most structurally similar to the NTP being used by the helicase for energy, the most likely mechanism is competition between the NTP and the drug. Furthermore, single amino acid substitution mutations in the NTP hydrolysis pocket, G288L and G288A, are shown to decrease the unwinding activity of nsp13 rather than confer resistance to nucleoside analogs. These results support nucleoside analogs as a treatment against multiple components of the SARS-CoV-2 replication machinery and suggest that nsp13 may have a high barrier to resistance with respect to these drugs.