SUMMARY The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to forming quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition, and should facilitate structure-based drug design strategies.

The mature capsids of human immunodeficiency virus type 1 (HIV-1) and other retroviruses are fullerene shells, composed of the viral CA protein, that enclose the viral genome and facilitate its delivery into new host cells1. Retroviral CA proteins contain independently-folded N-terminal and C-terminal domains (NTD and CTD) that are connected by a flexible linker2–4. The NTD forms either hexameric or pentameric rings, whereas the CTD forms symmetric homodimers that connect the rings into a hexagonal lattice3,5–13. We previously used a disulfide crosslinking strategy to enable isolation and crystallization of soluble HIV-1 CA hexamers11,14. By the same approach, we have now determined the X-ray structure of the HIV-1 CA pentamer at 2.5 Å resolution. Two mutant CA proteins with engineered disulfides at different positions (P17C/T19C and N21C/A22C) converged onto the same quaternary structure, indicating that the disulfide-crosslinked proteins recapitulate the structure of the native pentamer. Assembly of the quasi-equivalent hexamers and pentamers requires remarkably subtle rearrangements in subunit interactions, and appears to be controlled by an electrostatic switch that favors hexamers over pentamers. This study completes the gallery of sub-structures describing the components of the HIV-1 capsid and enables atomic level modeling of the complete capsid. Rigid-body rotations around two assembly interfaces appear sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone.

The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed hexameric arrays of the viral CA protein, but a high-resolution structure of the lattice has remained elusive. A three-dimensional map of two-dimensional crystals of the R18L mutant of HIV-1 CA was derived by electron cryocrystallography. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. Three important protein-protein assembly interfaces are required for capsid formation. Each CA hexamer is composed of an inner ring of six N-terminal domains and an outer ring of C-terminal domains that form dimeric linkers connecting neighboring hexamers. Interactions between the two domains of CA further stabilize the hexamer and provide a structural explanation for the mechanism of action of known HIV-1 assembly inhibitors.