Fig. 3

HIV IN SIM mutants decrease its own SUMOylation and Ubc9 binding. a IN is subject to SUMO3 modification. The 293 T cells were cotransfected with GFP-INwt and HA-SUMO3 or HA-SUMO3 alone. The SUMOylation of IN by HA-SUMO3 was detected by the SUMOylation assay as described in the Material and Methods. The SUMOylation level of IN is shown in the upper panel. Additionally, 2% of the total protein exacts were loaded onto SDS-PAGE gels to detect GFP-INwt and HA-SUMO3 expression (middle panel). Endogenous beta-actin was detected as a loading control (lower panel). b The IN 3VI mutant had higher SUMOylation levels than INwt. The 293 T cells were cotransfected with different plasmids as indicated, and the SUMOylation of GFP-INwt and 3VI was analyzed by using a SUMOylation assay. The SUMOylation level of INwt/mut is shown in the upper panel. The expression levels of GFP-INwt/mut and HA-SUMO3 are shown in the middle and the lower panels, respectively. c The interaction between IN and SUMO E2 protein Ubc9 was increased in IN SIMs mutant 3VI. HA-Ubc9 was cotansfected with GFP-INwt/3VI plasmids in 293 T cells and subjected to co-IP assay to study the binding affinity of IN-Ubc9. GFP-INwt/3VI bound HA-Ubc9 was shown in the upper panel. In addition, 2% of total cells were lysed to check the expression of GFP-INwt/3VI and HA-Ubc9 (middle and lower panels)