Structural basis of HIV-1 maturation inhibitor binding and activity

HIV-1 maturation inhibitors (MIs) interfere with the final step in the viral lifecycle by disrupting the ordered proteolytic processing of the viral Gag polyprotein into its individual domains. Bevirimat (BVM) and its analogs interfere with the final catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein (CA) C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. Here, we report atomic-resolution magic angle spinning (MAS) NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). BVM and IP6 can bind simultaneously to SP1, with BVM positioned in the center of its 6-helix bundle in a unique conformation. Importantly, the NMR-observed structural effects of BVM on IP6 binding suggest that the inhibitor stabilizes the 6-helix bundle in multiple ways. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our results reveal a novel allosteric mechanism by which BVM disrupts maturation and provide a structural explanation for BVM resistance as well as important guidance for the design of new MIs.