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Fig. 6 | Retrovirology

Fig. 6

From: HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

Fig. 6

Alanine mutations in Tat Ser-16 and Ser-46 reduced HIV-1 proviral DNA transcription replication. a Transcription activity of pNL4-3.Luc.R-E-vectors with WT and mutant Tat. 293T cells were transfected with the indicated proviral vectors and also co-transfected with EGFP expressing vector. At 48 h posttransfection, the cells were lysed and luciferase activity was detected. EGFP fluorescence was measured and used for normalization. Results are averages of quadruplicates from a typical experiment of 3 performed. Percent of activity relative to the WT Tat are shown above the bars. *p ≤ 0.001. b, c Replication of VSVg pseudotyped pNL4-3.Luc.R-E-vectors with WT and mutant Tat S16A, S16E, S46A and S46E sequences. 293T cells were transfected with proviral vectors and VSVg-expressing plasmid. At 48 h posttransfection, media was collected and used to infect CEM T cells (b) and for p24 measurement (c). In b, luciferase activity for WT, S16E and S46E viruses was normalized to p24. Percent of activity relative to the WT Tat is shown above the bars. *p ≤ 0.001. d Expression of Flag-tagged WT Tat and mutants. 293T cells were transfected with Flag-Tat vectors expressing WT Tat, Tat S16A, Tat S16D, Tat S46A and Tat S46D mutants. At 48 h posttransfection, the cells were lysed. The lysates were resolved on the 12% SDS-PAGE and immunoblotted with anti-Flag (upper panel) or anti-tubulin (low panel) antibodies. e 293T cells were transfected with vectors expressing EGFP, WT Tat-EGFP, Tat S16A-GEFP, Tat S16E-EGFP, Tat S46A-EGFP and Tat-S46E-EGFP. At 24 h posttransfection the cells were photographed on Olympus IX51 using a blue filter for EGFP fluorescence with × 300 magnification

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