Figure 3

Vpx exerts a general positive effect on lentiviral infection and results in an increased accumulation of full length viral DNA. A) Infections of DCs were carried out with VSVg-pseudotyped retroviral vectors bearing a CMV-GFP expression cassette (RVs, MOI 5) with or without Vpx-VLPs pre-incubation (MOI equivalent of 2, measured by exo-RT activity in comparison with standards of known infectivity). The percentage of infected cells was determined by flow cytometry 72 hours afterwards. B) DCs were pre-incubated with Vpx-VLPs at an MOI equivalent of 2 for 2 hrs prior to infection with a constant amount of HIV-1 (B), FIV (C) and MLV retroviral vectors (RV, D) at MOI 5. Cell aliquots were harvested at 4 and 24 hrs post-infection for HIV-1 and at 24 hrs only for FIV and MLV and analyzed by semi-quantitative PCR on serial five-fold sample dilutions (sample amount represented by triangles, as in the legend to Fig. 2), using primers that recognized specifically early and late products of reverse transcription. Amplification of actin DNA (actin) was used for normalization. For MLV, the positive control for 2LTRs amplification is represented by cell lysates of HeLa cells obtained 24 hrs post-infection with 10 fold less MLV vector than was used for DCs. PCR products were transferred onto a nylon membrane and hybridized with ³²P-labelled specific probes prior to phosphor imager analysis and quantification. One representative data set out of 3 to 4 independent experiments is shown here.