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

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 ␣-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-B 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.

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+T Cells

“…As the infected cells revert to a metabolically quiescent state, productive transcription of the integrated provirus is shut down (44). Transcription elongation is a major checkpoint in the HIV-1 life cycle, and it has been demonstrated that in order for the latent reservoir to be purged or reactivated, it will likely be essential to activate P-TEFb (10,44). Therefore, it is necessary to understand the ϩ T cells generated as described in Materials and Methods were cultured in the absence or presence of the proteasome inhibitor MG132 for 2 h or 4 h. Cell lysates were probed for the expression of Cyclin T1 (A), PPM1A (B), HSP90 (C), and anti-actin (␣-Actin) antibody (loading control) in immunoblots.…”

“…Studies have shown that various factors operating at the levels of transcription and posttranscription can restrict the expression of the integrated provirus in resting CD4 ϩ T cells (5,6). Transcriptional blocks to productive HIV-1 replication include epigenetic modifications at the viral long terminal repeat (LTR) and inadequate availability of activation-dependent transcription factors such as P-TEFb, NF-B, NFAT, Sp1, AP-1, and C/EBP (7)(8)(9)(10). Posttranscriptional factors regulating viral latency include inhibition of viral mRNA export to the cytoplasm and regulation of viral gene expression by cellular microRNAs (miRNAs) (11)(12)(13).…”

Perturbations de la transcription liées à une dérégulation de P-TEFb : cancer, Sida et hypertrophie cardiaque

“…Ainsi, tous les inhibiteurs de P-TEFb identifiés à ce jour inhibent efficacement la réplication du virus. Un taux restreint de P-TEFb actif, associé à la mise en place d'une structure chromatinienne restrictive au niveau du promoteur LTR, semble être à l'origine de l'établissement de la latence du virus dans les cellules T CD4 + primaires [22]. Dans les cellules dans lesquelles le VIH se réplique, l'équilibre entre les formes active et inactive de P-TEFb est modifié.…”

Progress and challenges in the use of latent HIV-1 reactivating agents

“…Latency happens at a very low frequency; only a small number of resting CD4 + T cells (approximately one million cells) harbor replication-competent latent provirus, which can be activated by mitogens [8] . Silencing of active infection in activated T cells and immediate silent integration in resting T cells both contribute to the establishment of latency, which has been demonstrated in vitro by several research groups [9][10][11][12] . The molecular mechanisms associated with latency are not completely understood [13][14][15][16] .…”

“…These can be categorized into the following groups, mainly based on pharmacological targets ( Figure 1): (1) histone deacetylase inhibitors (HDACi); (2) cytokines and chemokines; (3) DNA methyltransferase inhibitors (DNMTI); (4) histone methyltransferase inhibitors (HMTI); (5) protein kinase C (PKC) activators; (6) P-TEFb activators; and (7) unclassified agents, such as disulfram [9] . These compounds have been suggested as agents to reactivate HIV-1 and eradicate viral reservoirs.…”