The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids have been 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 performed in the Yeager laboratory. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. The refined pseudoatomic model corroborates existing biochemical, structural, and genetic data and revealed three interfaces required for capsid formation: i) The interactions within NTD-NTD hexamers are mediated by helices 1, 2 and 3, which associate as an 18-helix bundle in the center of the hexamer (as seen for the MLV CA_NTD hexamer), ii) The interactions between adjacent hexamers are mediated by CTD-CTD dimmers, specifically by parallel association of helix 9 from adjacent subunits (matching the crystallographic CACTD dimer), and iii) Intermolecular NTD-CTD interactions stabilize the capsid lattice and are formed by insertion of the NTD helix 4 into a groove in the CTD from an adjacent CA molecule (visualized for the first time). They inferred that two inhibitors, the 12-mer peptide CAI and the small molecule CAP-1, disrupt the intermolecular CA NTD-CTD interface, providing a structural explanation for the mechanism of action of these two HIV-1 assembly inhibitors.

Hexagonal Lattice of the HIV-1 CA Protein. (A) Overview of the hexagonal CA lattice, as viewed from the capsid exterior. Seven CA hexamers are shown, and the subunits are colored individually in two of the hexamers. Note that the CA N-terminal domains (NTD, bright shades) define the capsid exterior whereas the C-terminal domains (CTD, light shades) define the capsid interior. The CTD dimer (black dyad) and NTD-CTD (white asterisk) interfaces are highlighted here and are shown in expanded views in (B) and (C), respectively. (B) CA dimer viewed parallel (upper) and perpendicular (lower) to the two-fold axis of the CTD-CTD interface that connects adjacent hexamers. (C) NTD-CTD interface formed between adjacent subunits in each CA hexamer. Structures of bound CAI (yellow) and CAP-1 (green) illustrate how these inhibitors could disrupt interface formation and thereby block capsid assembly.