FIG 2.

HIV-1 viruses bearing changes on the capsid residue N57 are not able to infect nondividing cells. (A) PMA-treated (PMA) or untreated (Mock) THP-1–SAMHD1 knockout (KO) cells were challenged with increasing amounts of the indicated HIV-1-GFP viruses. Infection was determined by measuring the percentage of GFP-positive cells at 48 hpi. PMA-treated THP-1–SAMHD1 KO cells are used as a model of noncycling cells. (B) HIV-1-N57S/G208R recovers core stability during infection. Canine Cf2Th cells were challenged with the indicate viruses, and core stability was assayed by performing the fate-of-the-capsid assay. Eight hpi, cells were lysed and a postnuclear fraction (INPUT) was separated into SOLUBLE and PELLET fractions by using a 50% sucrose gradient, as described in Materials and Methods. INPUT, SOLUBLE, and PELLET fractions were analyzed for capsid using anti-p24 antibodies. PELLET represents the fraction of capsid that is forming cores. Experiments were performed at least three times, and a representative example is shown. (C to E) Human HT1080 (C), dog Cf2Th (D), or human HeLa (E) cells pretreated for 12 h with 0.5 μg/ml aphidicolin were challenged with increasing amounts of the indicated HIV-1 viruses. The use of 0.5 μg/ml of aphidicolin for 12 h stops the cell cycle at S phase, as determined by analyzing DNA content per cell using propidium iodide. Infection was determined by measuring the percentage of GFP-positive cells or the luciferase activity at 48 hpi, and standard deviations for triplicates are 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 (**), or <0.001 (***) or were nonsignificant. (F) PMA-treated (PMA) or untreated (Mock) U937 cells were challenged with increasing amounts of HIV-1-N57S/G208R-GFP viruses. Infection was determined by measuring the percentage of GFP-positive cells at 48 hpi. Experiments were performed at least three times, and a representative example is shown.