Figure 3

Differential effects of IN mutations within ₁₇₀ EHLK ₁₇₃ region on chromatin- and LEDGF-binding. A). Diagram of amino acids sequence and introduced mutations in HIV-1 IN ₁₇₀EHLK₁₇₃ domain. B). Chromatin binding profiles of IN double mutants within ₁₇₀EHLK₁₇₃. 293T cells were mock-transfected or transfected with equal amount of CMV-YFP-IN wild type or double mutants EH170,1AA, EK 170,3AA, HL171,2AA and HK171,3AA. At 48 h post-transfection, cells were fractionated into chromatin-bound and non-chromatin-bound fractions as described in Materials and methods. YFP-IN in each fraction was analyzed by IP and WB with anti-GFP antibody. Chromatin binding affinity was quantified by laser densitometry and results are shown as the percentage of chromatin-bound to total input of YFP-IN (lower panel). C) LEDGF-binding affinity within IN ₁₇₀EHLK₁₇₃ by co-IP assay. 293T cells were co-transfected with the SVCMVin-T7-LEDGF/p75 expressor and CMV-YFP-INwt/mut plasmid as indicated. After 48 h of transfection, 90% of cells were lysed and subjected to co-IP assay as described before. The upper panel showed the bound T7-LEDGF/p75 in each sample. 10% of cell lysates were used to detect the expression of YFP-INwt/mut and T7-LEDGF/p75 by WB using anti-GFP and anti-LEDGF antibodies respectively (middle panel and lower panel). D). LEDGF-binding affinity within IN ₁₇₀EHLK₁₇₃ detected by chemiluminescent co-IP assay. AcGFP1-INwt/mut or AcGFP1-C and ProLabel-LEDGF fusion proteins were coexpressed in 293T cells. After 48 h of transfection, cells were lysed and immunoprecipitated with anti-GFP antibody and the chemiluminescent signals from ProLabel-LEDGF present in the complexes were measured by using ProLabel Detection Kit II and valued as relative luminescence units (RLU). Results are representative of two independent experiments.