FIG 5.

Effects of PKR-mediated Tat phosphorylation on Tat activity. (A) Single mt (Ser/Thr to non-phospho amino acid Ala [A] or phospho-mimic amino acid Asp [D]) Tat activity in luciferase reporter assay. HeLa cells were cotransfected with each Flag-tagged wt or mt Tat plasmid (100 ng) and pLTR-luc reporter plasmid (200 ng), with pCMV-LacZ (20 ng) as a transfection control. Two days after transfection, luciferase activity was assessed in triplicates, and fold activities normalized with the transfection control are represented as means ± SD (n = 3). *, P < 0.05; **, P < 0.01 (mt versus wt Tat). (B and C) The activities of multiple A mutant (B) and D mutant (C) Tats were assessed by LTR-mediated luciferase reporter assay. (D) The sequence conservation ratio of each PKR target Ser/Thr site on HIV-1 Tat was assessed among 206 HIV-1 strains (101 strains from the United States, 30 from Europe, and 75 from Africa). Information on the 206 HIV-1 strains and their respective Tat amino acid sequences is shown in Table S4 in the supplemental material. (E) Alignment of HIV-1 Tat sequences of 6 African strains. Five PKR target sites, designated by yellow boxes, are mostly mutated with the non-phospho-form amino acids.