Tom Gallagher

Evolved SARS-CoV-2 variants of concern (VOCs) spread through human populations in succession. Major virus variations are in the entry-facilitating viral spike (S) proteins; Omicron VOCs have 29–40 S mutations relative to ancestral D614G viruses. The impacts of this Omicron divergence on S protein structure, antigenicity, cell entry pathways, and pathogenicity have been extensively evaluated, yet gaps remain in correlating specific alterations with S protein functions. In this study, we compared the functions of ancestral D614G and Omicron VOCs using cell-free assays that can reveal differences in several distinct steps of the S-directed virus entry process. Relative to ancestral D614G, Omicron BA.1 S proteins were hypersensitized to receptor activation, to conversion into intermediate conformational states, and to membrane fusion-activating proteases. We identified mutations conferring these changes in S protein character by evaluating domain-exchanged D614G/Omicron recombinants in the cell-free assays. Each of the three functional alterations was mapped to specific S protein domains, with the recombinants providing insights on inter-domain interactions that fine-tune S-directed virus entry. Our results provide a structure-function atlas of the S protein variations that may promote the transmissibility and infectivity of current and future SARS-CoV-2 VOCs. IMPORTANCE Continuous SARS-CoV-2 adaptations generate increasingly transmissible variants. These succeeding variants show ever-increasing evasion of suppressive antibodies and host factors, as well as increasing invasion of susceptible host cells. Here, we evaluated the adaptations enhancing invasion. We used reductionist cell-free assays to compare the entry steps of ancestral (D614G) and Omicron (BA.1) variants. Relative to D614G, Omicron entry was distinguished by heightened responsiveness to entry-facilitating receptors and proteases and by enhanced formation of intermediate states that execute virus-cell membrane fusion. We found that these Omicron-specific characteristics arose from mutations in specific S protein domains and subdomains. The results reveal the inter-domain networks controlling S protein dynamics and efficiencies of entry steps, and they offer insights on the evolution of SARS-CoV-2 variants that arise and ultimately dominate infections worldwide.