Production of different single-cycle replicating viruses and their infection in HeLa-CD4-CCR5-β-Gal cells. A). To evaluate the trans-incorporation of RT and IN in VSV-G pseudotyped viral particles, viruses released from 293T cells transfected with NLlucΔBglΔRI provirus alone (lane 6) or cotransfected with different Vpr-RT-IN expressors and a VSV-G expressor (lane 1 to 5) were lysed, immunoprecipitated with anti-HIV serum. Then, immunoprecipitates were run in 12% SDS-PAGE and analyzed by Western blot with rabbit anti-IN antibody (middle panel) or anti-RT and anti-p24 monoclonal antibody (upper and lower panel). B) The infectivity of trans-complemented viruses produced in 293 T cells was evaluated by MAGI assay. HeLa-CD4-CCR5-LTR-β-Gal cells were infected with equal amounts (at 10 cpm/cell) of different IN mutant viruses and after 48 hours of infection, numbers of β-Gal positive cells (infected cell) were monitored by X-gal staining. Error bars represent variation between duplicate samples and the data is representative of results obtained in three independent experiments.