Figure 1

CA subunits virions with engineered cysteines at the NTD-CTD capsid interface spontaneously crosslink into hexamers in HIV-1 particles. (A) A view of the NTD-CTD interface showing the NTD helices 4 and 7 (blue) from one subunit in close proximity to the CTD helices 8 and 11 (red) from another subunit (PDB: 3H4E). Residues mutated to cysteines for crosslinking are shown in orange and are connected by green dotted lines. The two endogenous Cys are shown for comparison. Distances in angstroms (Å) from a Cβ-carbon of one amino acid residue on NTD1 of one subunit to a Cβ-carbon on a second amino acid residue on CTD2 from a different subunit are shown. The Cβ-Cβ distance between endogenous cysteines is shown for comparison. (B, C, and D) Immunoblot analysis of viral lysates. Pelleted particles were dissolved in 1X SDS sample buffer in the absence (B) or presence (C) of β-mercaptoethanol. Samples were then separated by SDS-PAGE and analyzed by immunoblotting with CA-specific antibody. (D) Analysis of the single mutants M68C and E212C under non-reducing conditions.