Lenacapavir (LEN) is a first-in-class capsid (CA) inhibitor for the treatment and prevention of HIV-1 infection. While LEN has shown potent antiviral activity across all major HIV-1 subtypes, the impact of existing HIV-1 CA sequence diversity on the activity of LEN remains to be determined. Here, we identified natural polymorphisms within the LEN-binding site and assessed each for their impact on viral infectivity and susceptibility to LEN. Using a co-crystal structure of LEN in complex with a CA hexamer, we identified 29 binding site residues within five angstroms of LEN and analyzed each for naturally occurring polymorphisms across a multiclade collection of >
10,000 unique HIV-1