FIG 3.

Infection of nondividing cells by HIV-1-N57S is stopped after reverse transcription but prior to nuclear translocation. (A) PMA-treated THP-1–SAMHD1 knockout (KO) cells were challenged with the indicated DNase-pretreated HIV-1-GFP viruses. (Upper) Infection was determined by measuring the percentage of GFP-positive cells by flow cytometry at 48 hpi. (Lower) In parallel, cells from similar infections were lysed at 7 hpi and total DNA extracted. The DNA samples collected at 7 hpi postinfection were used to determine the levels of late reverse transcripts by real-time PCR. As a control, we used 10 μM the reverse transcription inhibitor nevirapine (Nev). Late reverse transcript levels were normalized to actin. (B) Similarly, HeLa cells pretreated with 0.5 μg/ml aphidicolin for 12 h were subsequently infected by the indicated DNase-pretreated HIV-1–Luc viruses. (Upper) Infection was determined by measuring luciferase activity at 48 hpi. In parallel, cells from similar infections were lysed at 7 and 24 hpi, and total DNA was extracted. The DNA samples collected at 7 hpi were used to determine the levels of late reverse transcripts by real-time PCR. (Lower) HIV-1 2-LTR circles, a marker for nuclear import, were quantified by real-time PCR of DNA samples collected at 24 hpi. In addition, integration was measured by Alu-PCR in DNA samples collected at 24 hpi. The levels of late reverse transcripts, 2-LTR circle, and products of Alu-PCR were normalized to actin. Nevirapine was used as a control. Experiments were repeated at least three times, and a representative example is shown. Results were analyzed using two-tailed Student's t test. Differences were considered statistically significant at a P value of <0.05 (*), <0.01 (**), <0.001 (***), or <0.0001 (****) or were nonsignificant (ns).