Molecular mechanism of ziresovir targeting the fusion glycoprotein of respiratory syncytial virus

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and the elderly worldwide. Although prophylactic monoclonal antibodies and RSV vaccines are available for preventing severe RSV infection, unmet medical need remains for an effective antiviral agent to treat patients who do not benefit from these interventions. Ziresovir (formerly AK0529) is a potent, selective, and orally bioavailable RSV fusion inhibitor with proved antiviral efficacy and clinical benefits. To understand the molecular mechanism of action, we computationally modeled ziresovir with the RSV fusion (F) protein. Here, we present a cryo-EM structure of the RSV F protein-ziresovir complex, elucidating the molecular interactions underlying the drug binding, revealing ziresovir specifically binds to the central cavity within the metastable prefusion conformation of RSV F protein. Leveraging this structural insight, we engineered site-directed RSV mutants guided by both the cryo-EM binding model and drug-resistant RSV variants for fusion inhibitors identified in vitro , and demonstrated that these resistant viruses do not replicate as efficient as wild-type RSV and indicated a fitness cost for viral escape from drug treatment. Collectively, these findings unveil the structural mechanism of ziresovir-mediated viral inhibition, providing a framework for developing the next-generation RSV fusion inhibitors.Author summaryRSV is a common respiratory virus and can cause serious lung infections, especially in infants, older adults, and people with serious medical problems. Preventive strategies, including prophylactic monoclonal antibodies and RSV vaccines, have been developed to mitigate severe RSV infections, a critical unmet medical need persists. There are no orally administered antiviral agents for RSV infection that are clinically available globally. Ziresovir which is a potent, selective, and orally bioavailable RSV fusion inhibitor has been proven antiviral activity and confirmed clinical benefits. In this study, we test antiviral activity of ziresovir against RSV clinical isolates, which suggests that ziresovir have broadly activity against clinical isolates. Although, we assessed viral fitness for six ziresovir treatment-emergent F subsitutions and test susceptibility to ziresovir of these ziresovir treatment-emergent F substitutions contained recombinant RSV variants. To elude the structure basis for these F substitutions against ziresovir treatment, we determined the cryo-EM structure of the complex formed by the RSV F trimer protein and ziresovir that performed to explicitly elucidate the binding mode of ziresovir to F protein. We further found that mutations in key residues at the binding interface not only alter the binding affinity between ziresovir and the F protein but also impair the antiviral efficacy of ziresovir. Collectively, our work clarifies the molecular mechanism underlying the interaction between ziresovir and F protein, and provides a structural basis for the rational design of next-generation anti-RSV inhibitors.