Establishment of HIV Latency in Primary CD4(+) Cells Is due to Epigenetic Transcriptional Silencing and P-TEFb Restriction

Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4960, USA.
Journal of Virology (Impact Factor: 4.44). 07/2010; 84(13):6425-37. DOI: 10.1128/JVI.01519-09
Source: PubMed


The development of suitable experimental systems for studying HIV latency in primary cells that permit detailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viral eradication strategies. Primary CD4(+) T cells were isolated from peripheral blood and amplified by antibodies to the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reporters and either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells and reamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on the H80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent protein marker expression; however, more than 95% of these latently infected cells could be reactivated after stimulation of the TCR by alpha-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analogously to Jurkat T cells, latent proviruses in primary CD4(+) T cells are enriched in heterochromatic markers, including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there was recruitment of NF-kappaB to the promoter and conversion of heterochromatin structures present on the latent provirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected primary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, which can be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIV long terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency in primary CD4(+) T cells.

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    ABSTRACT: Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. Copyright © 2015 Elsevier Inc. All rights reserved.
    Virology 09/2015; 483. DOI:10.1016/j.virol.2015.03.036 · 3.32 Impact Factor
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    • "cytic cell lines as well as in CD4 þ resting T cells . We found that IR treatment of cells correlated with dissocia - tion of the histone deacetylase HDAC1 , and methyl transferase SUV39H1 from the HIV - 1 LTR promoter . Direct involvement of these factors in HIV - 1 latency was shown previously by a number of colleagues ( Pearson et al . , 2008 ; Tyagi et al . , 2010 ) . Our data further suggest a triggering effect of IR on HIV - 1 transcription via epigenetic mechanisms . In fact , the studies of the last decade revealed involvement of various epigenetic factors in IR stress - induced transcription activation associated with DNA damage response . In IR - sensitive tumor cells , the γ - IR has been "
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    Virology 07/2015; 485:1-15. DOI:10.1016/j.virol.2015.06.021 · 3.32 Impact Factor
    • "These latently infected cells form a viral reservoir, forcing patients to remain on lifelong suppressive therapy. The prevailing view (Coffin and Swanstrom, 2013; Siliciano and Greene, 2011) holds that proviral latency results from HIV transcription being controlled by the host-cell activation state (i.e., environment) since relaxation of activated lymphocytes to a resting-memory state is correlated with increased epigenetic silencing of the HIV promoter and increased cytoplasmic sequestration of transcription factors that activate HIV transcription (Pearson et al., 2008; Tyagi et al., 2010). In this model, HIV infects activated T cells, which allow active viral replication, and if these cells ''relax'' to resting-memory T cells, which generally restrict HIV infection, viral latency ensues (Figure 1, left). "
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