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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：
10439610
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/10439610
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 Persons 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.

项目总结/摘要目前的抗HIV-101疗法可以预防艾滋病的发展，但不能治愈感染。记忆性CD 4 + T细胞作为转录沉默的前病毒，免疫系统无法检测到，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心的三种蛋白质来激活HIV-11前病毒的转录大量CD 4 + T细胞中HUSH复合物的破坏增加了HIV-11的转录， HIV-11传播感染的前病毒和动力学，表明HUSH复合体起主导作用尽管如此，对单个克隆的检查显示了对HIV-11前病毒沉默反应的异质性， HUSH破坏，以及与已知沉默因子如SETDB 1不一致的相关性。确定HUSH复合物沉默HIV-11前病毒的要求。携带HIV-HUS 1的前病毒表现出一系列HUSH反应性，筛选以鉴定区分具有不同HUSH表型的克隆的宿主沉默因子。将独立表征对原病毒转录和原病毒染色质特征的影响。我们希望这些实验能更好地了解HUSH是如何被招募并维持转录的，目的二是研究HUSH复合物在CD 4 + T细胞中的作用， HUSH复合物的破坏激活了某些细胞中LINE-B1的表达，行提出了关于HUSH复杂破坏的可能后果的问题。全球转录和将对原代人CD 4 + T细胞进行染色质分析，其中HUSH复合物组分预期特定逆转录转座子的表达增加，但也预期免疫相关基因的表达增加。 HUSH复合物活性的检测，以及可用于监测细胞中HUSH复合物活性的标志物。在丢失的ATAC-Seq峰内的转录因子基序将有助于鉴定DNA结合蛋白， HUSH复合物也将在脐带血人CD 34+造血干细胞中被破坏。这些实验将告诉我们，HUSH细胞是否能在小鼠体内重建免疫系统。复合物是人类造血系统发育所必需的，尤其是对CD 4 + T细胞的发育所必需的。将评估HUSH复合物对体内HIV-HIV-1潜伏期的贡献。将用从HIV-HIV 1+细胞中收获的CD 4 + T细胞检查HIV-HIV 1前病毒再活化的失活这些研究预期将有助于提高对HIV-11转录调控机制的理解，有助于阐明HIV-11转录调控的机制，给定前病毒的状态，开发在临床背景下破坏HIV-11前病毒的新方法，更广泛地说，增加了对基因调控的基本理解。