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.

项目概要/摘要目前的抗 HIV-1 疗法可以预防艾滋病进展，但无法治愈长期存在的 HIV-1 感染。记忆 CD4+ T 细胞作为转录沉默原病毒，免疫系统无法检测到，并且因此，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 (HUSH)复合物破坏大量CD4+T细胞中的HUSH复合物增加了HIV-1的转录。原病毒和 HIV-1 传播感染的动力学，表明 HUSH 复合体起着主导作用尽管如此，对各个克隆的检查显示出对 HIV-1 原病毒沉默的异质性 HUSH 干扰，以及与已知沉默因素（例如 SETDB1）不一致的相关性。目标 1 将是确定 HIV-1 原病毒 HUSH 复合物沉默的要求。 CD4+ T 细胞克隆组。携带表现出一系列 HUSH 反应性的 HIV-1 前病毒的人将遭受功能丧失筛选以识别区分具有不同 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。我们希望通过这些实验更好地了解 HUSH 是如何被招募并维持转录的 HIV-1 原病毒的沉默目标 2 将检查 HUSH 复合物在 CD4+ T 细胞中的作用。转录和发育。HUSH 复合物的破坏会激活某些细胞中的 LINE-1 表达。线路引发了有关 HUSH 复合体破坏可能产生的后果的问题。染色质分析将在人类原代 CD4+ T 细胞上进行，其中含有 HUSH 复合物成分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。丢失的 ATAC-Seq 峰中的转录因子基序将有助于识别招募的 DNA 结合蛋白 HUSH 复合物也会在脐带血人类 CD34+ 造血干细胞中被破坏。用于重建小鼠免疫系统的细胞这些实验将告诉我们HUSH是否有效。复合物对于人类造血发育或特定的 CD4+ T 细胞 Aim 3 至关重要。将评估 HUSH 复合物对体内 HIV-1 潜伏期的贡献 HUSH 复合物的作用。将使用从 HIV-1+ 收获的 CD4+ T 细胞检查 HIV-1 原病毒重新激活的灭活情况预计这些研究将来自接受抗 HIV-1 抑制治疗的个体和人源化小鼠。提高对 HIV-1 转录调控机制的理解，帮助预测转录特定原病毒的状态，开发在临床环境中破坏 HIV-1 原病毒的新方法，更一般地说，增加对基因调控的基本理解。