Lenacapavir treatment–emergent HIV-1 capsid resistance mutations are frequently associated with replication defects

Lenacapavir (LEN) is a long-acting HIV-1 capsid inhibitor that binds to the HIV-1 capsid protein with picomolar antiviral activity, disrupting its function and inhibiting viral replication. Here, we identified capsid mutations in samples from individuals treated with LEN across two clinical trials that were considered potential LEN resistance-associated mutations. The <i>gag</i> encoding regions of clinical isolates with capsid mutations, as well as associated site-directed mutants, were cloned into the infectious molecular clone pXXLAI and pNL4-3-JRFL-secNLuc, encoding replication-competent HIV-1. Their effects on LEN susceptibility, replication kinetics, and three-dimensional capsid structure were investigated. Phenotypic analyses of the HIV-1 clinical isolates and site-directed mutants revealed that all resistance-associated mutations decreased LEN susceptibility to various degrees but were frequently associated with substantial replication defects. Structural modeling confirmed that LEN binding in the binding pocket was altered in the presence of capsid mutations, with predicted binding affinity changes correlating with observed potency shifts. These findings provide insights into LEN-resistance mechanisms and underscore the unusually high fitness costs associated with treatment-emergent capsid mutations.