Data underlying Figs 1–5 and S2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is adapting to continuous presence in humans. Transitions to endemic infection patterns are associated with changes in the spike (S) proteins that direct virus-cell entry. These changes generate antigenic drift and thereby allow virus maintenance in the face of prevalent human antiviral antibodies. These changes also fine tune virus-cell entry dynamics in ways that optimize transmission and infection into human cells. Focusing on the latter aspect, we evaluated the effects of several S protein substitutions on virus-cell membrane fusion, an essential final step in enveloped virus-cell entry. Membrane fusion is executed by integral-membrane “S2” domains, yet we found that substitutions in peripheral “S1” domains altered late-stage fusion dynamics, consistent with S1-S2 heterodimers cooperating throughout cell entry. A specific H655Y change in S1 stabilized a fusion-intermediate S protein conformation and thereby delayed membrane fusion. The H655Y change also sensitized viruses to neutralization by S2-targeting fusion-inhibitory peptides and stem-helix antibodies. The antibodies did not interfere with early fusion-activating steps; rather they targeted the latest stages of S2-directed membrane fusion in a novel neutralization mechanism. These findings demonstrate that single amino acid substitutions in the S proteins both reset viral entry—fusion kinetics and increase sensitivity to antibody neutralization. The results exemplify how selective forces driving SARS-CoV-2 fitness and antibody evasion operate together to shape SARS-CoV-2 evolution.