FIGURE 2.

HIV-1 IN is SUMOylated by overexpressed or endogenous SUMO proteins in the cell. A, 293T cells were co-transfected with plasmids encoding FLAG-tagged IN_WT, or IN_3KR, or IN_3EQ and Ubc9; and His-tagged SUMO-1, or SUMO-2, or SUMO-3 or an empty vector (mock) and, 40 h later, were lysed in denaturing conditions followed by purification on nickel-NTA beads. B, 293T cells co-expressing WT or 3KR or 3EQ IN-FLAG and His-tagged ubiquitin were treated as in A. C, 293T cells expressing His-IN_WT or His-IN_3R were treated as in A, and modification by endogenous SUMO-1 or SUMO-2/3 was assessed. A longer exposure time of the inset enclosed in the black square is shown (middle). The arrows indicate SUMO-1-conjugated IN forms. D, acetylated forms of His-IN_WT or His-IN_3R were analyzed following purification as in A. Proteins expressed in the cells or enriched on nickel-NTA beads in A–D were visualized by Western blot with the indicated antibodies. WB, Western blot; WCL, whole cell lysate.