Effects of lethal phenotype-defective IN mutants on VSV-G-pseudotyped HIV-1 infection. 293T cells were co-transfected with the RT/IN/Env-deleted NL4.3Luc/DBg/DRI provirus, the VSV-G expressor and different Vpr-RT-IN expressors. After 48 hrs of transfection, viral particles were harvested and concentrated by ultracentrifugation. A. Equal amounts of viral particles were lysed and loaded on an SDS-PAGE and analyzed by anti-HIV WB. B. To assess viral infection, equal amounts of virions were used to infect the CD4+ C8166 T cell line. At different time points, equal cell numbers were collected and the HIV infection was evaluated by luciferase assay. C. 12 hrs post-infection, 1x106 C8166 cells were lysed and the total viral DNA from each sample was analyzed by HIV specific PCR and visualized in 1% agarose gel. The positive control was loaded in lane 14. D. To test the functional complementation of IN mutants, different combinations of Vpr-RT-IN mutant expressors, as indicated, were co-transfected with NL4.3Luc/DBg/DRI provirus and VSV-G expressor in 293T cells. After 48 hrs of transfection, viral particles were collected, and used to infect dividing C8166 cells. At 72 hrs post-infection and the cell-associated luciferase activity was measured. NC: negative control. The results are representative of three independent experiments.