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

SARS-CoV-2 variants bearing complex combinations of mutations have been associated with increased transmissibility, COVID-19 severity, and immune escape. S:D614G may have facilitated emergence of such variants since they appeared after S:D614G 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 S:D614. In all cases, infectivity of the revertants was compromised. Rare SARS-CoV-2 lineages that lack S:D614G were identified and the infectivity of these was dependent upon S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. All three mutations, S:Q613H, S:D614G, and S:H655Y, stabilized Spike on virions, consistent with selection of these mutations by a common molecular mechanism. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y 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 S:D614G and S:H655Y 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 S:D614G or S:H655Y spontaneously mimic the smFRET signal that ACE2 induces in the parental molecule. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y 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. ### Competing Interest Statement The authors have declared no competing interest.