Figure 3

QT-PCR analysis of late reverse transcripts generated in HeLa/CD4 and NP2/CD4/CXCR4 cells challenged with restricted and non-restricted MLV-B and MLV-NB viral vectors. MLV-B and MLV-NB cored viral vectors pseudotyped with the different Envs described in Figure 1 were used to challenge confluent monolayers of HeLa/CD4 and NP2/CD4/CX/CR4 cells in 12 well trays with MOIs of 0.25-1. Controls for infection included incubation of infected cells for 5 minutes at 4°C or trypsinising target cells (to strip cell surface receptors off) just prior to infection. Normal infection involved incubating cell cultures O/N (12-16 hrs) to 48 hrs at 37°C (for the FACS equivalent). Total extracted DNA was normalised to 250 or 500 ng/PCR reaction. Primers and probes were designed to amplify the eGFP target, with an internal genomic GAPDH control. The data shown have been normalised to the GAPDH amplification and for the background level of transcripts seen in uninfected cell controls for each experiment. (a) (i) MLV-B viral vectors pseudotyped with VSV-G Env, MLV-NB viral vectors pseudotyped with HIV-2 MCN Env, cells were incubated 5 minutes at 4°C and overnight 37°C incubation, data shown are for RT transcripts detected after overnight 37°C incubation minus the number detected for the 5 minutes at 4°C incubation for each Env, MOI of 1 (ii) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i); (b) (i and ii) HIV-1 and HIV-2 MCN pseudotyped MLV-NB CA, titrated at MOIs 0.25-1 (iii and iv) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i and ii); (c) MLV-NB viral vectors pseudotyped with GALV and Ampho Envs, cells were incubated O/N, MOI of 0.3. Data shown for the QT-PCRs are representative of 3 or more independent experiments. Error bars represent +/- STDEV within 1 experiment.