No 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...

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.