Figure 4
Vif-induced cell cycle arrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3e-n-GFP e-f+r+ (A and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP+ cells was examined by flow cytometry using DRAQ5 at 24 and 42 hours post-infection as previously described [4]. The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cell cycle model and the ratio was plotted [4]. All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55, 57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54–56], and mouse-anti-β-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.