Uncovering genetic determinants of attenuation and drug resistance in SARS-CoV-2 and the development of a novel papain-like protease inhibitor

The continued circulation of SARS-CoV-2 underscores the urgent demand for novel antiviral strategies. Among emerging variants, Omicron BA.1—harboring over 50 amino acid substitutions—displays markedly reduced pathogenicity compared to the early Washington (WA1) isolate. Deciphering the molecular basis of this attenuation is key to guiding future therapeutic development. While the over 30 spike protein mutations have been implicated, they do not fully explain BA.1’s attenuated phenotype, pointing to additional contributions from non-spike changes. To test this, I engineered recombinant viruses incorporating BA.1-derived non-spike substitutions and compared their replication dynamics with isogenic WA1. Our results reveal that both broad non-spike mutations and specific changes in nsp6 drive the attenuated phenotype. Notably, nsp6 variants were associated with recovery of enhanced lipid droplet accumulation in infected or transfected cells compared to wild-type nsp6, suggesting impaired viral modulation of lipid metabolism as a potential mechanism for reduced replication capacity of BA.1. Another pressing issue is antiviral resistance, particularly against nirmatrelvir, the FDA-approved, oral protease inhibitor targeting SARS-CoV-2 main protease (Mpro, nsp5). Mapping resistance pathways is essential for therapeutic durability. Using recombinant viruses carrying Mpro variants identified through mutational screening, I determined that substitutions at residues S144, E166, and H172 confer viral resistance to nirmatrelvir. Although some mutations reduced viral fitness, compensatory mutations emerged, restoring replication efficiency and reinforcing the adaptability of the virus under drug pressure. To overcome this challenge, additional drug targets must be pursued. The papain-like protease (PLᵖʳᵒ) is a critical viral enzyme with therapeutic potential, though historically difficult to inhibit. Our collaborators developed a series of noncovalent inhibitors optimized for binding to the Val70 side chain and BL2 groove pocket of PLᵖʳᵒ. The lead candidate, Jun12682, demonstrated potent activity against SARS-CoV-2 and its variants—including strains resistant to nirmatrelvir—in both cell culture and animal models. Oral administration of Jun12682 significantly improved survival and reduced pulmonary viral burden and pathology in infected mice, highlighting PLᵖʳᵒ inhibitors as a promising class of next-generation oral antivirals.