Primary CD4+ T cells from hCyclin T1 mice support efficient reverse transcription of HIV-1.
(A) Kinetics of early reverse transcription products in mouse T cells. MACS purified CD4+ T cells from three individual hCyclin T1 mice were activated for 36 h by anti-CD3/28. Cells were then spin infected with benzonase-treated VSVG-pseudotyped HIV-GFP (MOI = 2). 3 h post infection, cells were washed to remove virus and replated on anti-CD3/28. At different time points, cells were collected, washed and frozen down. DNA was isolated by phenol extraction and Real Time PCR analysis was preformed. To show the de novo formation of reverse transcription products, AZT (15 µM) was added at different time points and cells were collected as above starting from the next time point (Dashed lines). Real Time-PCR was performed using primers and probes specific for 18S rRNA (for cell number) and R-U5 (for early reverse transcription product). The copy number of reverse transcription product was then normalized to cell number and displayed as copy number per cell. (B) Kinetics of late reverse transcription products. Samples from panel A were subjected to Real Time-PCR using primers and probes specific for U5-Gag. (C, D) Activated CD4+ T cells from three mice were infected and analyzed for early (panel C) and late reverse transcription (panel D) products as described in panel A. For comparison, PHA activated human PBMC CD4+ T cells from three donors were infected and analyzed in parallel. All values represent the arithmetic means ±SD. Data represent two independent experiments, each with cells from three human donors or three mice.