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Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

Significance Respiratory syncytial virus (RSV) causes major disease in pediatric and elderly patients, urging the development of efficacious therapeutics. This study establishes a recombinant RSV reporter strain for drug discovery and identifies an entry inhibitor class targeting the viral fusion (F) protein. Biochemical, structural, and functional characterization of the inhibitor spotlights two microdomains governing the conformational stability of prefusion F. Mutations in these domains cause broad cross-resistance against the compound and RSV entry inhibitors in preclinical and clinical development, without mandatory loss of in vivo pathogenicity, challenging the possible clinical benefit of current RSV entry inhibitor classes. Anti-RSV campaigns should better target postentry steps or proactively circumvent resistance to entry inhibition. The resistant RSV reporter strain developed here establishes a strategy toward this goal. Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.