Virus infectivity is decreased by mutations in the Psi region. (A) Viral infectivity was determined using a reporter cell line expressing the gfp gene under the control of the HIV-2 LTR (Ghost CCR5/CXCR4). Equivalent amounts of VSV-G pseudotyped wild type (WT) and mutant HIV-2, normalised on RT activity, were used to infect 5 × 103 Ghost CCR5/CXCR4 cells and expression of GFP was measured at 72 h post-infection by FACS. Data from at least three independent experiments are plotted. Error bars represent the SD. (B) Virus inputs were adjusted according to the DM virus packaging defect as to infect the Ghost cells with an equivalent level of genomic RNA for WT and DM virus. Top panel: HIV-2 genomic RNA was extracted from two set amounts of virions used to infect the Ghost cells and viral RNA levels were assessed by semi-quantitative RT-PCR using neat, 1:10 and 1:100 dilution of the RNA sample. A GAPDH carrier RNA was added during the extraction and amplified in parallel by RT-PCR to control for RNA loss during the RNA extraction (not shown). A control without RT was performed on 10 μl of neat RNA (-). Bottom panel: The percentage of GFP positive cells was measured by FACS at 72 h post-infection. Arbitrary levels of RNA of 1 and 4 are equivalent to 62.5 cpm and 250 cpm of WT virus, respectively, as measured by RT activity assay. Data from four independent experiments are shown on the chart. Error bars represent the SD. *, Student t test p value < 0.05; **, p value < 0.01.