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CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency.

ABSTRACT: The establishment of HIV proviral latency requires the creation of repressive chromatin structures that impair the initiation of transcription and restrict RNAP II elongation. We have found that C-promoter binding factor-1 (CBF-1), a CSL (CBF-1, Su(H) and Lag-1)-type transcription factor and key effector of the Notch signaling pathway, is a remarkably potent and specific inhibitor of the HIV-1 LTR promoter. Knockdown of endogenous CBF-1 using specific small hairpin RNAs expressed on lentiviral vectors results in the partial reactivation of latent HIV proviruses, recruitment of RNAP II, loss of histone deacetylases and the concomitant acetylation of histones. An important property of any repressor utilized to establish HIV latency is that it must become displaced or deactivated upon T-cell activation. Consistent with this hypothesis, CBF-1 mRNA and protein levels are highest in quiescent or unstimulated T cells but decline rapidly in response to proliferative stimulation such as activation of the T-cell receptor or treatment with TNF-alpha. We conclude that CBF-1 is a previously overlooked factor that induces transcriptional silencing during the establishment of HIV latency.

SUBMITTER: Tyagi M

PROVIDER: S-EPMC2140115 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency.

Tyagi Mudit M Karn Jonathan J

The EMBO journal 20071115 24

The establishment of HIV proviral latency requires the creation of repressive chromatin structures that impair the initiation of transcription and restrict RNAP II elongation. We have found that C-promoter binding factor-1 (CBF-1), a CSL (CBF-1, Su(H) and Lag-1)-type transcription factor and key effector of the Notch signaling pathway, is a remarkably potent and specific inhibitor of the HIV-1 LTR promoter. Knockdown of endogenous CBF-1 using specific small hairpin RNAs expressed on lentiviral v ...[more]

PMID: 18007589

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Project description:BackgroundThe regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized.MethodsTo better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes.ResultsIn activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues.ConclusionsCycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.

Project description:BackgroundCurrent antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs).ResultsJ-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP- and GFP+ cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators.ConclusionThe active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.

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CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency.

Publications

CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency.

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