CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.