Figure 1
The function of Vpx in the infection of DCs is conserved uniquely in members of the SIV SM /HIV-2 lineage. A) HIV-2 and SIVMAC LVs rely on Vpx for the infection of DCs. VSVg-pseudotyped SIVMAC and HIV-2 LVs (coding or not for Vpx) were produced in 293T cells, purified by ultracentrifugation and used to infect DCs at a multiplicity of infection (MOI) of 3. Vpx-containing SIVMAC VLPs (Vpx-VLPs) similarly produced were added onto DCs at MOI equivalent of 2 (as measured by exo-RT test with standards of known infectivity) for 2 hrs prior to infection with the above-mentioned LVs. GFP+ cells were scored 3 days later by flow cytometry. B) Only Vpx proteins from the SIVSM/HIV-2 lineage rescue the infectivity defect of Vpx-deficient SIVMAC LVs. VSVg-pseudotyped SIVMAC LVs (SIV15-, coding gag-pro-pol) were produced in presence or absence of Flag-tagged Vpx proteins derived from SIVMAC, SIVRCM and HIV-2. Virions were then normalized for their infectious titer on HeLa cells and used to infect DCs at MOI 0.3 or 3. The incorporation of Flag-Vpx proteins into virion particles was assessed by Western blot (right panel). The different migration on SDS-PAGE of HIV-2 and SIVMAC Vpx proteins has already been reported [40]. C) Only Vpx proteins from the SIVSM/HIV-2 lineage increase WT HIV-1 LVs infection in a pre-incubation assay. Non-infectious SIVMAC VLPs containing the different Flag-Vpx proteins were produced and used as described in A in a pre-incubation assay to test their effect on WT HIV-1 LVs infectivity (used at a constant MOI of 3). Incorporation of Flag-Vpx proteins into VLPs was assessed by Western blot (right panel). One representative data set out of 3 to 5 independent experiments is shown for each panel.