PMC:3320587 / 31873-37949 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":"NF-κB and NFAT5 binding play independent activating roles in MTb-mediated induction of HIV-1 replication\nWe next examined the functional impact of specifically disrupting NF-κB or NFAT5 binding on regulation of HIV-1 replication by infecting bulk PBMC from four normal donors with the wild-type or mutant HIV-1Lai/Bal-env molecular clones. After overnight infection, cells were co-infected with MTb (CDC1551) or mock-infected and free virus levels were detected by measuring p24 levels in culture supernatants at days 4, 7, and 12 post-HIV-1 infection.\nReplication of each mutant virus was reduced in comparison to wild-type virus in mock- and MTb-co-infected cells at all timepoints examined (Figures 4A–4B). As depicted in the histograms displayed in Figure 4C, at day 12 post-HIV-1 infection in MTb-co-infected cells there was a significant reduction in levels of HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, and HIV-1Lai/Bal-env -N5-Mut in comparison to wild-type virus (Figure 4C). In the absence of MTb co-infection, p24 levels were also significantly lower in the PBMC cultures infected with HIV-1Lai/Bal-env–κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut and HIV-1Lai/Bal-env-N5-Mut, but not HIV-1Lai/Bal-env-κB I-Mut, in comparison to wild-type virus (Figure 4C). None of the mutations completely abolished viral induction by MTb co-infection. However, the replication of each mutant virus was impaired to a more significant extent in the context of MTb co-infection, consistent with important roles for both NF-κB and NFAT5 in MTb-induced HIV-1 replication.\n10.1371/journal.ppat.1002620.g004 Figure 4 MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR.\nSpecific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb. PBMC from four normal donors were infected with 1000 TCID50 HIV-1Lai/Bal-env-WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli). Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures. Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p\u003c0.05; **, p\u003c0.01, as compared to HIV-1Lai/Bal-Env-WT. To specifically examine the functional impact of NF-κB and NFAT5 binding site usage in MDM, which are efficiently infected by both pathogens, we next isolated MDM from four normal donors and infected these cells with 1000 TCID50 of wild-type HIV-1Lai/Bal-env or with the mutant viral clones HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut. After overnight incubation, cell cultures were co-infected with MTb (CDC1551) or left infected with virus alone. Supernatants were collected at days 4, 7, and 12 post-HIV-1 infection and virus replication was measured. As shown in Figures 5A and 5C, in the absence of MTb co-infection, replication of mutant viral molecular clones (HIV-1Lai/Bal-env-κB I-Mut (p\u003c0.05), HIV-1Lai/Bal-env-κB II-Mut (p\u003c0.01), and HIV-1Lai/Bal-env-κB I+II-Mut (p\u003c0.01) as well as HIV-1Lai/Bal-env-N5-Mut (p\u003c0.01) were significantly reduced in comparison to the wild-type virus (Figures 5A–5C).\n10.1371/journal.ppat.1002620.g005 Figure 5 Disruption of NF-κB or NFAT5 sites in the LTR inhibits MTb-induced HIV-1 replication in MDM.\nMDM from four normal donors were infected with 1000 TCID50 of HIV-1Lai/Bal-env–WT or the mutants HIV-1Lai/Bal-env-κB I-Mut, HIV-1Lai/Bal-env-κB II-Mut, HIV-1Lai/Bal-env-κB I+II-Mut, or HIV-1Lai/Bal-env-N5-Mut, and were either (A) left infected with virus alone or (B) co-infected with MTb CDC1551 (1∶1 MDM∶bacilli). Viral p24 levels in the culture supernatants were measured at days 4, 7 and 12 post-infection. Presentation of viral p24 levels at day 12 in cultures infected with (C) HIV-1 alone or (D) co-infected with MTb are shown as histograms. (*, p\u003c0.05; **, p\u003c0.01, as compared to HIV-1Lai/Bal-env–WT). When the cultures were co-infected with MTb, p24 levels increased when the cells were infected with either the wild-type or, to lower levels, with the κB-I mutant virus (Figure 5B). However, p24 levels were significantly inhibited in cells infected with HIV-1Lai/Bal-env -N5-Mut (p\u003c0.01) (Figure 5D). Moreover, p24 levels were significantly lower at day 12 in cultures infected with HIV-1Lai/Bal-env-κB II-Mut and HIV-1Lai/Bal-env-κB I+II-Mut as compared to infection of cells with wild-type virus (Figure 5D). Notably, although replication of the mutant virus with the single, proximal NF-κB binding site mutation in the shared NFAT5/NF-κB element (HIV-1Lai/Bal-env-κB I-Mut) was diminished, this was not significant at day 12 (Figures 5B–5D).\nWe note that overall p24 levels were noticeably lower in the cultures co-infected with MTb compared with those infected with HIV-1 alone. This is consistent with previous observations showing that MTb infection of human primary macrophage cultures ex vivo suppresses HIV-1 infection due to chemokine synthesis and the enhanced expression of cellular inhibitory factors [48]–[50]. Given that NF-κB is efficiently activated in primary MDM in response to TLR agonists [51] and as we have shown in Figure 2, NFAT5 gene expression is also induced by TLR agonists, MDM are a suitable experimental system to analyze the effect of NF-κB versus NFAT5 binding site mutations on virus replication in isolated MDM in the absence or presence of MTb co-infection. Taken together, the results from PBMC and MDM co-infection experiments demonstrate that the conserved NFAT5 binding site plays as important a transcriptional role in LTR regulation by MTb as do the NF-κB sites.","divisions":[{"label":"title","span":{"begin":0,"end":104}},{"label":"p","span":{"begin":105,"end":552}},{"label":"p","span":{"begin":553,"end":1600}},{"label":"figure caption","span":{"begin":1601,"end":2647}},{"label":"title","span":{"begin":1645,"end":1732}},{"label":"p","span":{"begin":1733,"end":2647}},{"label":"p","span":{"begin":2648,"end":3623}},{"label":"figure 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