Recurrent and novel evolutionary pathways drive in vitro HIV-1 lenacapavir resistance with diverse phenotypic consequences.

The small-molecule inhibitor lenacapavir (LEN) has firmly established the HIV-1 capsid as a promising clinical target. However, early studies have shown that HIV-1 can develop resistance to LEN by acquiring capsid mutations. These mutations are restricted to the drug-binding pocket, but the breadth of the virus's capacity to evolve under LEN-directed pressure remains incompletely understood. Here, we employ an accelerated evolution approach to probe HIV-1's capacity for LEN resistance. Despite strong sequence conservation across thousands of clinical strains, two positions within the binding pocket exhibit high mutational tolerance and diverse resistance profiles. These low-level resistance mutations recurrently select the high-level resistance mutation M66I and together with further mutations, synergistically enhance viral fitness by restoring nuclear entry. Subsequently, multiple charge-reversing substitutions distal to the binding pocket emerge. Located at inter-hexamer interfaces, these substitutions modulate capsid stability and mitigate LEN's capsid-disrupting effects. A highly resistant variant carrying the E213K substitution becomes drug dependent; it requires LEN to generate infectious particles. As the novel pathways observed here may represent natural extensions of recurrent ones, this work supports surveillance and forecasting efforts while guiding further drug development to sustain the capsid as a viable antiviral target.