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 were first detected after S:D614G had gone to fixation. Consistent with the hypothesis that variant emergence was contingent upon S:D614G, the infectivity of SARS-CoV-2 variants was shown here to be dependent upon S:D614G. SARS-CoV-2 lineages that propagated in the absence of S:D614G were found to be dependent upon either S:Q613H or S:H655Y. S:Q613H, S:D614G, and S:H655Y each stabilized Spike on virions but were dispensable in the absence of S1/S2 cleavage. CryoEM and a single molecule assay for RBD conformation revealed that S:D614G and S:H655Y shift the RBD towards the open conformation required for ACE2-binding and therefore on pathway for infection. These experiments indicate 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, and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.