FIG 4.

Functional aspects of virus particle components. (A) The virus particle fractions of HIV-1 pseudotypes (WT, D116G, ΔIN, Y15A, Y15F, Y15H, and Y15W) containing 10 ng of p24 were subjected to endogenous RT assays. The amount of cDNA product was determined by performing qPCR with R1-25/AA55 primer pairs. Values are shown as percentages relative to the average value for the WT. All graphs present the mean results ± standard deviations (n ≥ 3). Reactions using WT virus fraction without dNTP and mock-transfected HEK 293T culture supernatant (293T sup) were used as controls. (B) In situ uncoating assays were performed as described previously (47). Briefly, HeLa cells were spinoculated with fluorescently labeled WT and mutant viruses containing GFP-Vpr or S15-dTomato for 2 h. At various times after the spinoculation, the total number of complexes that entered and fused into the cytoplasm (Vpr-GFP⁺, S15-dTomato⁻), the number of CA-coated complexes (Vpr-GFP⁺, CA-Cy-5⁺), and the number of CA-uncoated complexes that lost CA staining (Vpr-GFP⁺, CA-Cy-5⁻) were counted. Representative images of GFP-Vpr (green), S15-Tomato (red), and HIV-1 CA (blue) and a merged image of these three colors taken at the 1-h time point are shown (upper). An upward arrow shows red, green, and blue triple fluorescent colocalization. An arrowhead shows green and blue double fluorescent colocalization. A downward arrow shows green fluorescence only. The data were graphed at each time point as the percentage of cytoplasmic coated particles (Vpr-GFP⁺, CA-Cy-5⁺) among the total number of the fused particles (Vpr-GFP⁺, S15-dTomato⁻). For BafA-treated samples, the percentage of Vpr-GFP⁺ and CA-Cy-5⁺ complex among the total number of Vpr-GFP⁺ particles at the 4-h time point is shown. Data at each time point represent the mean result ± the standard deviation (n = 4).