Figure 3. CA domain mutants affect Gag clustering on membranes.
(a) Schematic of a hexagonal lattice showing the Gag CA domain at the two-, three- and six fold symmetry contacts, as arranged in the immature Gag lattice in HIV-1 assembly sites and immature virions (Schur et al., 2015) (PDB entry 4USN used in panel a–d). (b) Model of the CA domain at the two fold, with mutated residues in “Gag two-fold” marked in red. (c–d) As (b), for Gag mutated at the three- and six-fold symmetry contacts, respectively. (e) Confocal micrograph of GUVs, 10 min after adding 100 nM Gag-ATTO594. Membrane in red and Gag in white/cyan. (f) As (e), using Gag-ATTO594 two-fold. GUVs with diffuse Gag binding are denoted with white asterisks. (e–f) White arrows mark Gag clusters on GUVs. Scale bar, 10 µm. (g) Type of Gag binding to GUVs. For each protein, GUVs were counted in 10 confocal z-stacks, and classified according to having no Gag fluorescence, only clustered Gag fluorescence (black), or a diffuse Gag fluorescence covering the entire membrane (with or without additional brighter clusters, gray). All measurements were conducted on the same preparation of GUVs, with error bars indicating the standard deviation between three repeats conducted on separate GUV preparations. *significant at p<0.05 level by Student's t-test.