Figure 1

IID-IN mutants map to surface of IN crystal structures and exhibit defects in binding to INI1 in vitro: A. A cartoon illustrating the positions of IID-IN mutations on three domains of IN (not drawn to scale). NTD = N-terminal domain; CCD = Central core domain; CTD = C-terminal domain. Sequence spanning the mutant residues from HIV-1 and three other related lentiviruses are indicated below the cartoon, and the parent residues that are mutated is shown in red. B. Mutant residues with high surface accessibility are highlighted on Pymol rendition of two domain crystal structures of IN. The left diagram illustrates the structure of a dimeric core + C-terminal domains (PDB: 1EX4); and the right diagram illustrates the tetramer formed by the core + N-terminal domains (PDB: 1K6Y). Residues are indicated in the following colors: D202- green, K111- blue, Q137- red, K71- cyan. Monomers within the IN dimer and tetramer are represented by different colors. C. IID-IN mutants are defective for binding to INI1 in vitro. In vitro GST pull-down assays were performed by using 293 T cell lysates expressing YFP-WT IN or YFP-IID-IN mutants. Lysates were normalized for YFP-IN (WT or IID-IN mutant) expression and bound to GST-INI1 immobilized onto glutathione sepharose beads. GST-INI1 input was measured by Coomassie stain. WB = Western blot. D. Graphic representation of degree of binding of each YFP-IID-IN mutant relative to that of YFP-WT, as measured by densitometric analysis. Amount of YFP-IN bound was normalized against amount of YFP-IN loaded.