Fig. 3.

Analysis of viral block in TE and H2 cell lines. (A) Clones were infected at different multiplicities of VSV-HIV-Puro virus. Total DNA was extracted 7 days after infection and copy number of integrated provirus determined by qPCR. Proviral copy number was normalized per 100 ng of total DNA. Data are representative of 3 independent experiments; error bars represent the standard error. (B) (Left) Analysis of viral mRNA expression. Cytoplasmic mRNA was extracted 7 days after VSV-HIV-Neo infection at the indicated multiplicities. First strand cDNA synthesis and amplification of the target DNA was performed by qPCR, using primers recognizing the neomycin reporter gene. Results were normalized to copies of viral mRNA per copy of GAPDH. (Right) Resistance to infection by HIV-Neo was assessed in parallel. Neomycin was added to the medium 48 h after infection, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results shown are typical of those obtained in 3 independent experiments. (C) (Left) Nuclear and cytoplasmic RNA was extracted 7 days after infection with 10-fold decreasing dilutions of VSV-HIV-TK. After first strand cDNA synthesis, TK and GAPDH cDNA was amplified by PCR. (Right) TE and H2 cells were infected at an MOI of 3 × 10⁻² with HIV-Puro. Seven days after infection; nuclear RNA was extracted and quantified by qRT-PCR, using primers recognizing the puromycin reporter gene. Results are relative to values of GAPDH and the ratio found in TE cells was taken as reference. All data are representative of 3 independent experiments. Errors are standard error of the mean. HI, heat-inactivated.