Article

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

ABSTRACT

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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    • "Latency is a complex process that involves several factors, including epigenetic modification (Williams et al. 2006; Easley et al. 2010), promoter occlusion (Duverger et al. 2009), a lack of necessary transcription factors in resting cells (Williams and Greene 2007), and restriction of translation by viral and host miRNAs (Huang et al. 2007). In latent CD4 + resting T cells, the lack of active forms of key cellular transcription factors (Nabel and Baltimore 1987; Bohnlein et al. 1988; Kinoshita et al. 1997; West et al. 2001; Ganesh et al. 2003; Shan et al. 2011), and of the HIV Tat protein and its cellular cofactors (Selby and Peterlin 1990; Jones and Peterlin 1994; Tyagi et al. 2010), limits viral transcription (Lassen et al. 2004; Williams and Greene 2007; Shan et al. 2011). Additionally, DNA methylation and histone modifications have been postulated to promote transcriptional silencing of integrated proviruses (Coull et al. 2000; Williams et al. 2006; Blazkova et al. 2009; Kauder et al. 2009). "
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    • "For many years, the absence of a viable evolutionary fitness argument bolstered the notion that HIV proviral latency was a bystander effect (i.e., epiphenomenon) ineludibly resulting from transcriptional silencing during relaxation of activated lymphocytes to a quiescent-memory state (Figure 2a). However, despite the vast literature of associative evidence linking latent HIV integration sites to silenced chromatin and correlating latency with cellular silencing[32,33], there was also evidence for an alternate model where latency was controlled by viral gene-regulatory circuitry[9,20,34]without strict dependence on cellular state (Figure 2a). Critically, the hypothesis that latency establishment was driven by cellular state had never been directly tested (i.e., lymphocytes had never been infected and tracked in real time as they underwent relaxation to memory). "
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    • "ChIP analysis was performed as previously described (Tyagi et al., 2010). Briefly, (1 Â 10 8 ) THP-1 cells were infected by the virus carrying pNL4-3-ΔE-EGFP. "
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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.
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