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The HUSH complex in HIV-1 latency

HIV-1潜伏期中的HUSH复合体

基本信息

批准号：
10176390
负责人：
JEREMY LUBAN
金额：
$ 78.33万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10176390
关键词：
ATAC-seq Acquired Immunodeficiency Syndrome Anti-HIV Agents CD34 gene CD4 Positive T Lymphocytes Cell Culture Techniques Cell Line Cells Chromatin Clinical Clone Cells Complementary DNA Complex DNA DNA Binding DNA-Binding Proteins Development Evaluation Excision Exhibits Foundations Gene Expression Regulation Gene Silencing Genes Genetic Transcription Goals HIV-1 Harvest Hematopoietic Hematopoietic stem cells Heterogeneity Human Human Chromosomes Immune Immune system Individual Infection Integration Host Factors Kinetics Monitor Mus Pharmaceutical Preparations Phenotype Phylogeny Play Primate Lentiviruses Proteins Proviruses Reporting Resistance Retrotransposon Role SETDB1 gene Stimulus Transcriptional Regulation Umbilical Cord Blood Ursidae Family Viral Viremia Virus antiretroviral therapy experimental study genetic element humanized mouse improved in vivo loss of function memory CD4 T lymphocyte mouse model novel strategies preservation prevent reconstitution recruit response tool transcription factor transcriptome sequencing vpr Gene Products

项目摘要

Project Summary/Abstract Current anti-HIV-1 therapies prevent progression to AIDS but do not cure infection. HIV-1 persists in long-lived memory CD4+ T cells as a transcriptionally silent provirus, where it is undetectable by the immune system, and therefore resistant to extirpation. Recently, we reported that primate immunodeficiency virus Vpx and Vpr proteins activate HIV-1 provirus transcription by degrading the three proteins of the human silencing hub (HUSH) complex. Disruption of the HUSH complex in bulk CD4+ T cells increased transcription from HIV-1 proviruses and kinetics of HIV-1 spreading infections, indicating that the HUSH complex plays a dominant role in HIV-1 provirus silencing. Nonetheless, examination of individual clones showed heterogeneity in response to HUSH disruption, and inconsistent correlation with known silencing factors such as SETDB1. Aim 1 will be to identify requirements for HUSH complex silencing of the HIV-1 provirus. Sets of CD4+ T cell clones bearing HIV-1 proviruses that exhibit a range of HUSH responsiveness will be subjected to loss-of-function screens to identify host silencing factors that distinguish clones with different HUSH phenotypes. Such factors will be characterized independently for effects on provirus transcription and provirus chromatin features. From these experiments we expect to better understand how HUSH is recruited to, and maintains transcriptional silencing of, HIV-1 proviruses. Aim 2 will be to examine the role of the HUSH complex in CD4+ T cell transcription and development. Disruption of the HUSH complex activates LINE-1 expression in certain cell lines raising questions about possible consequences of HUSH complex disruption. Global transcription and chromatin profiling will be performed on primary human CD4+ T cells in which HUSH complex components are disrupted. Increased expression of particular retrotransposons is expected, but also immune-related genes of relevance to HIV-1, and markers that may be used to monitor HUSH complex activity in cells. Examination of transcription factor motifs within lost ATAC-Seq peaks will aid identification of DNA-binding proteins that recruit the HUSH complex. The HUSH complex will also be disrupted in cord blood human CD34+ hematopoietic stem cells used to reconstitute an immune system in mice. These experiments will tell us whether the HUSH complex is essential for human hematopoietic development generally or for CD4+ T cells specifically. Aim 3 will assess the contribution of the HUSH complex to HIV-1 latency in vivo. The effect of HUSH complex inactivation on HIV-1 provirus reactivation will be examined with CD4+ T cells harvested from HIV-1+ individuals on anti-HIV-1 suppressive therapy and from humanized mice. These studies are expected to improve mechanistic understanding of HIV-1 transcriptional regulation, help prognosticate the transcriptional status of a given provirus, develop new approaches for disrupting the HIV-1 provirus in the clinical context, and, more generally, increase fundamental understanding of gene regulation.

项目摘要/摘要目前的抗 HIV-1 疗法可以防止进展为艾滋病，但不能治愈感染。 HIV-1 持续存在于长寿者体内记忆 CD4+ T 细胞作为转录沉默的原病毒，免疫系统无法检测到它，并且因此能够抵抗灭绝。最近，我们报道了灵长类免疫缺陷病毒 Vpx 和 Vpr 蛋白质通过降解人类沉默中枢的三种蛋白质来激活 HIV-1 原病毒转录（嘘）复杂。破坏大量 CD4+ T 细胞的 HUSH 复合物会增加 HIV-1 的转录原病毒和 HIV-1 传播感染的动力学，表明 HUSH 复合体发挥着主导作用 HIV-1 原病毒沉默。尽管如此，对各个克隆的检查显示出对反应的异质性 HUSH 中断，以及与已知沉默因素（例如 SETDB1）不一致的相关性。目标 1 是确定 HIV-1 原病毒 HUSH 复合物沉默的要求。 CD4+ T 细胞克隆组携带 HIV-1 原病毒并表现出一系列 HUSH 反应性的人将遭受功能丧失筛选以确定宿主沉默因子，以区分具有不同 HUSH 表型的克隆。这些因素将独立表征对原病毒转录和原病毒染色质特征的影响。从 these experiments we expect to better understand how HUSH is recruited to, and maintains transcriptional HIV-1 原病毒的沉默。 Aim 2 will be to examine the role of the HUSH complex in CD4+ T cell 转录和发展。 Disruption of the HUSH complex activates LINE-1 expression in certain cell lines raising questions about possible consequences of HUSH complex disruption. 全球转录和 chromatin profiling will be performed on primary human CD4+ T cells in which HUSH complex components are 扰乱了。预期特定反转录转座子的表达增加，而且免疫相关基因的表达也增加与 HIV-1 的相关性，以及可用于监测细胞中 HUSH 复合物活性的标记物。检查 transcription factor motifs within lost ATAC-Seq peaks will aid identification of DNA-binding proteins that recruit HUSH 情结。 The HUSH complex will also be disrupted in cord blood human CD34+ hematopoietic stem cells used to reconstitute an immune system in mice. These experiments will tell us whether the HUSH complex is essential for human hematopoietic development generally or for CD4+ T cells specifically. 目标 3 将评估 HUSH 复合物对体内 HIV-1 潜伏期的贡献。 HUSH 复合物的功效 inactivation on HIV-1 provirus reactivation will be examined with CD4+ T cells harvested from HIV-1+ individuals on anti-HIV-1 suppressive therapy and from humanized mice. 这些研究预计将 improve mechanistic understanding of HIV-1 transcriptional regulation, help prognosticate the transcriptional status of a given provirus, develop new approaches for disrupting the HIV-1 provirus in the clinical context, 更一般地说，增加对基因调控的基本理解。