S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness (preprint)

ABSTRACT

SARS-CoV-2 variants bearing complex combinations of mutations have been associated with increased transmissibility, COVID-19 severity, and immune escape. SD614G may have facilitated emergence of such variants since they appeared after SD614G had gone to fixation. To test this hypothesis, Spike sequences from an immunocompromised individual with prolonged infection, and from the major SARS-CoV-2 variants of concern, were reverted to the ancestral SD614. In all cases, infectivity of the revertants was compromised. Rare SARS-CoV-2 lineages that lack SD614G were identified and the infectivity of these was dependent upon SQ613H or SH655Y. Notably, Gamma and Omicron variants possess both SD614G and SH655Y, each of which contributed to infectivity of these variants. All three mutations, SQ613H, SD614G, and SH655Y, stabilized Spike on virions, consistent with selection of these mutations by a common molecular mechanism. Among sarbecoviruses, SQ613H, SD614G, and SH655Y are only detected in SARS-CoV-2, which uniquely possesses a polybasic S1/S2 cleavage site. Results of genetic and biochemical experiments here demonstrated that SD614G and SH655Y are likely adaptations to the cleavage site. CryoEM revealed that both mutations shift the Spike receptor binding domain towards the open conformation required for ACE2-binding and Spikes bearing either SD614G or SH655Y spontaneously mimic the smFRET signal that ACE2 induces in the parental molecule. Data from these orthogonal experiments demonstrate that SD614G and SH655Y are convergent adaptations to the polybasic S1/S2 cleavage site, which stabilize S1 on the virion in the open RBD conformation that is on-pathway for target cell fusion, and thereby act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.