Compensatory substitutions in the HIV-1 capsid reduce the fitness cost associated with resistance to a capsid-targeting small-molecule inhibitor.

UNLABELLED: The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes. IMPORTANCE: The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.