Epigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into Latency

Abstract

The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP؉ and d2EGFP ؊ cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP ؉ cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes IB␣ and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-␣ associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.The vast majority of human immunodeficiency virus (HIV) infections result in active viral transcription and replication; however, in a few rare cases the virus can enter a long-lived latent state in which viral gene expression is silenced. Although the pool of latently infected cells is very small (approximately 1 in 10 6 resting CD4 ϩ T cells in the peripheral circulation), the ability to latently infect cells helps HIV to establish chronic infections despite strong humoral and cellular immune responses and to evade intensive antiretroviral therapy. Siliciano and his colleagues (29, 41) have proposed that the generation of latently infected cells is a consequence of normal maturation and cellular differentiation events leading to the formation of quiescent T-cell populations. For example, if an activated effector T cell becomes infected by HIV when it is in the process of reverting to being a resting memory T cell, a stably integrated but transcriptionally silent form of the provirus can be generated. Similarly, Zack and colleagues have effectively used the HIV SCID-hu (Thy/Liv) mouse model to recapitulate the generation of latent HIV during thymopoiesis (1, 4, 5). As infected CD4 ϩ CD8 ϩ thymocytes differentiate...

Loss of the Brm-Type SWI/SNF Chromatin Remodeling Complex Is a Strong Barrier to the Tat-Independent Transcriptional Elongation of Human Immunodeficiency Virus Type 1 Transcripts

“…These observations suggest that the lack of expression of the Brm-type SWI/SNF complex would be one of the major causes of the attenuated HIV-1 proviral expression observed in resting CD4 ϩ T cells. In the presence of Tat, stochastic expression patterns of HIV were observed previously by several groups (8,19,(32)(33)(34). For example, it was previously reported by Weinberger et al that Tat stochastics appear to be sufficient to generate phenotypic bifurcation (32).…”

Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

“…Host transcription factors (TFs) such as nuclear factor kappa light-chain enhancer of activated B cells (NF-B) have multiple binding sites in the 5= long terminal repeat (LTR) of the HIV-1 genome, and their binding has been demonstrated to be necessary to initiate HIV-1 transcription (6). Sequestration of these TFs in the cytoplasm is one of the mechanisms enabling viral latency (7).…”

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

“…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).…”