Figure 6

Imp7 KD inhibits viral DNA but not viral RNA accumulation in the nuclei. (A) HeLa cells were infected with a DNAse-treated and purified HIV-1 vector and incubated for 2 hours at 4°C and then 4–6 hours at 37°C. Infected cells were fractionated into nuclear and (a) cytoplasmic fractions and nucleic acids extracted, purified and divided into two aliquots. One aliquot was treated with RNAse A, re-purified and used for DNA quantification. The other aliquot was treated with RNAse-free DNAse and used for first-strand cDNA synthesis. Cyclophillin A cDNA was then amplified by PCR in each fraction to examine cross-contamination of nuclear fractions with cytoplasmic material and the overall efficiency of first-strand cDNA synthesis. Cyt, cytoplasmic fractions; Nu, nuclear fractions; W, wild-type virus; M, mutant virus; RT-, cytoplasmic fraction with no RT during first-strand cDNA synthesis; ctr-, primers only; Mw, GeneRuler 100 bp DNA molecular weigh marker. The band migrating at approximately 500 bp is cyclophilin A, lower molecular weigh bands are PCR artefacts. The experiments were performed using 10 fold serial dilutions of the cDNA mix. (B) HIV-1 RNA accumulates in the nuclei shortly after infection. HeLa cells were infected at an MOI of approximately 0.2 with a VSV-G pseudotyped HIV-1 vector (wild type) or with the same amount (p24 normalized) of vector with a mutation in RT and unable to reverse transcribe (RT-). Cells were incubated for 2 hours at 4°C and then 4 hours at 37°C following which samples were fractionated in nuclear and cytoplasmic fractions and treated as described in (A). Taqman PCR was used to measure the amount of viral DNA and RNA in each fraction. First-strand cDNA synthesis reactions carried out in the absence of RT gave undetectable signal. Values shown are average values ± SD of triplicate experiments. Similar results were obtained in two independent experiments. (C) Accumulation of HIV-1 DNA is reduced in imp7 KD cells. HeLa DxR or imp7 KD cells were infected with the same dose of a VSV-G pseudotyped HIV-1 vector, incubated 2 hours at 4°C and then 6 hours at 37°C, following which nuclear and cytoplasmic extracts were prepared and treated as described in (A). After first-strand cDNA synthesis, Taqman PCR was used to measure the amount of viral DNA and RNA in each fraction. First-strand cDNA synthesis reactions carried out in the absence of RT gave undetectable signal. Values shown are average copy number of viral RNA or DNA/μg total nucleic acids ± SD of triplicate experiments. Similar results were obtained in two independent experiments.