PMC:3320587 / 15343-19325 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3320587","sourcedb":"PMC","sourceid":"3320587","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3320587","text":"MTb increases HIV-1 LTR activity of HIV-1 subtypes B, C, and E\nTo compare the functional impact of MTb stimulation on subtype-specific HIV-1 LTR activity, we first constructed reporter plasmids containing viral subtype B, C, and E LTRs (−208 to + 64 nt relative to the transcription start site) linked to the firefly luciferase reporter gene. After transfection of the monocytic THP-1 cell line with these plasmids, cells were stimulated with an irradiated whole cell lysate of MTb (H37Rv). We note that MTb lysate induces inflammatory responses in monocytes that resemble those induced in response to live MTb (see for example, [35]–[37]). Upon stimulation, the B, C, and E LTR-driven reporters demonstrated a significant enhancement in luciferase activity (Figure 1A) and the magnitude of this effect was subtype-specific. Subtype C LTRs displayed the strongest activity, while the LTRs from subtype E isolates consistently showed the weakest activity (Figure 1A), consistent with previous studies demonstrating subtype-specific LTR activity that used TNF as a stimulus [38], [39].\n10.1371/journal.ppat.1002620.g001 Figure 1 NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.\n(A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E. HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3. THP-1 cells (0.8×106/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the Renilla luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours. Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures. In the histogram, open bars represent individual LTR activities in untreated cells. Light grey bars represent mean values of LTR activities from each subtype in untreated cells. Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate. LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures. Results are from three independent experiments performed in duplicate (*, p\u003c0.05; **, p\u003c0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment. THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1Lai LTR and an HIV-1Lai LTR containing the NFAT5 binding site mutations (N5-Mut). After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C. Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p\u003c0.01). LTR activity was also suppressed in the untreated cells but to a lesser extent (p\u003c0.05). Results are from three independent experiments performed in duplicate and adjusted to Renilla luciferase control expression (*, p\u003c0.05; **, p\u003c0.01). Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells. THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C. Whole cell extracts were collected and analyzed by western blot with anti-NFAT5 antibody. An antibody directed against Lamin-B1 was used as a loading control. The histogram shows densitometric analysis of the NFAT5 bands from the western blot autoradiograph displayed and values represent mean band intensities at 0, 8, and 24 hours post-stimulation with MTb 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