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. 2019 Dec 17;10(6):e02591-19. doi: 10.1128/mBio.02591-19

FIG 5.

FIG 5

HIV-1 Tat exhibits AHR-dependent recruitment of P-TEFb complex for subsequent phosphorylation of RNA Pol II. (A and B) AHR forms complexes with HIV-1 Tat, cyclin T1, and ARNT. (A) HEK293T cells were transfected with pRK-Flag/tat plasmid for 24 h, cell lysates were subjected to immunoprecipitation (I.P.) by using anti-AHR-specific antibody or an IgG control, and immunoblotting was performed by using the indicated antibodies. (B) The anti-CycT1-specific antibody was used for immunoprecipitation. (C) AHR knockdown impaired Tat-mediated recruitment of P-TEFb. HEK293T cells with or without AHR knockdown with siRNAs were transfected with pRK-Flag/tat plasmid for 24 h. Immunoprecipitation was performed with anti-CycT1-specific antibody, and immunoblotting was performed by using the indicated antibodies. (D and E) AHR knockdown decreased the phosphorylation of RNA Pol II and the binding of phosphorylated RNA Pol II to the HIV-1 5ˊ-LTR. HEK293T cells with or without AHR knockdown were infected with HIV-luc/VSV-G for 24 h, the phosphorylation of RNA Pol II at the serine 2 of CTD domain was measured with Western blotting using specific antibody (D), and the association of phosphorylated RNA Pol II with HIV-1 5ˊ-LTR was measured by ChIP-PCR assay using nuc1- and nuc2-specific primers (E). (F to H) Ligand treatment enhanced AHR nuclear translocation, association with the DHS1 region of 5ˊ-LTR, and phosphorylation of RNA Pol II. HEK293T cells were infected with HIV-luc/VSV-G for 24 h and then treated with Kyn (200 μM), FICZ (100 nM), or DMSO (0.1%) for 30 min. Cells were collected, and the cytosol and nucleus components were fractionated and immunoblotted with the indicated specific antibodies (F), ChIP-PCR assay was conducted using DHS1-specific primers to measure the association of AHR with HIV-1 5ˋ-LTR (G), and the whole-cell lysates were used to detect the phosphorylation of RNA Pol II (H). Results are representative of three independent experiments. **, P < 0.01; ***, P < 0.001 (significant difference in an unpaired t test). CycT1, cyclin T1.