Supplemental Data
Files in this Data Supplement:
- Supplemental figure 3 (.pdf, 179 KB) - Figure S3. IN SUMOylation sites and multimerization. (A) The representation of IN tetramer was created with Polyview-3D, using the coordinates (PDB ID: 1K6Y). Each of the four protomers of IN (NTD and CCD) is shown in a different color (purple, blue, green and yellow). For each monomer the side chains of residues K46, E48, K136, E138 are shown in red. (B) The image of IN dimer was generated as in (A) using the coordinates (PDB ID: 1EX4). Each monomer (CCD and CTD) is shown in a different color (green and blue) and the side chains of residues K136, E138, K244, E246 are shown in red.
- Supplemental figure 2 (.pdf, 83 KB) - Figure S2. Impairment of SUMO conjugation to IN requires simultaneous substitution of candidate K residues. 293T cells expressing Flag-tagged WT or mutant IN along with Ubc9 and His-tagged SUMO1, SUMO2 or SUMO3 were lysed in denaturing conditions. Proteins recovered on Ni-NTA beads were detected with an anti-IN antibody.
- Supplemental figure 1 (.pdf, 77 KB) - Figure S1. Analysis of sequence conservation of candidate SUMOylation sites in IN from primate lentiviruses. Structure-based amino acid sequence alignment of HIV-1 (K03455), SIVcpz (AF115393), HIV-2rod (M15390) and SIVmac (AY588946). Secondary structure elements (��, alpha helix; ��, beta strand; ��, 310 helix; T, turn) are shown above (PDB ID: 1K6Y) and below (PDB ID: 1EX4) the structure. Residue numbering corresponds to HIV-1 IN sequence HXB2. Direct and reverse SUMO consensus motifs are highlighted within red and green squares, respectively. Generated with Multialin and ESPRIT.
- Supplemental figure 4 (.pdf, 156 KB) - Figure S4. HIV-1 IN is SUMOylated in virus producing-cells. 293T cells stably expressing His-SUMO-2 or WT were co-transfected with the pNL4-3delEnvGFP plasmid and a vector encoding the VSVg envelope to generate HIV-1 viral stocks. Forty hours after transfection, virus-producing cells and viral particles concentrated by ultracentrifugation were subject to purification in denaturing conditions over Ni-NTA agarose beads. Proteins of interest were detected by Western blot with antibodies against IN or SUMO-2/3.