The HUSH complex in HIV-1 latency
The HUSH complex in HIV-1 latency
批准号:
10176390
负责人:
JEREMY LUBAN
金额:
$78.33万
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-07-01 至 2024-06-30
关键词:
ATAC-seqAcquired Immunodeficiency SyndromeAnti-HIV AgentsCD34 geneCD4 Positive T LymphocytesCell Culture TechniquesCell LineCellsChromatinClinicalClone CellsComplementary DNAComplexDNADNA BindingDNA-Binding ProteinsDevelopmentEvaluationExcisionExhibitsFoundationsGene Expression RegulationGene SilencingGenesGenetic TranscriptionGoalsHIV-1HarvestHematopoieticHematopoietic stem cellsHeterogeneityHumanHuman ChromosomesImmuneImmune systemIndividualInfectionIntegration Host FactorsKineticsMonitorMusPharmaceutical PreparationsPhenotypePhylogenyPlayPrimate LentivirusesProteinsProvirusesReportingResistanceRetrotransposonRoleSETDB1 geneStimulusTranscriptional RegulationUmbilical Cord BloodUrsidae FamilyViralViremiaVirusantiretroviral therapyexperimental studygenetic elementhumanized mouseimprovedin vivoloss of functionmemory CD4 T lymphocytemouse modelnovel strategiespreservationpreventreconstitutionrecruitresponsetooltranscription factortranscriptome sequencingvpr Gene Products
中文摘要
项目概要/摘要
目前的抗HIV-11抗体疗法可以预防艾滋病的发展,但不能治愈感染. HIV-1在长期存活的
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,
因此对根除具有抗性。 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
(嘘)复杂。 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
在HIV-11前病毒沉默中。尽管如此,对单个克隆的检查显示了对以下物质的反应的异质性:
HUSH破坏,以及与已知沉默因子如SETDB 1的不一致相关性。目标1是
确定HUSH复合物沉默HIV-11前病毒的要求。 CD 4 + T细胞克隆集
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。等因素
将独立表征对原病毒转录和原病毒染色质特征的影响。从
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,
沉默HIV-11前病毒。 目的二是研究HUSH复合物在CD 4 + T细胞中的作用。
转录和发展。HUSH复合物的破坏激活某些细胞中的LINE-B1表达
对HUSH综合体中断可能造成的后果提出了质疑。 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
被打乱了 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。
与HIV-HUS 1的相关性,以及可用于监测细胞中HUSH复合物活性的标志物。 审查
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 Complex。HUSH复合物也将在脐带血人CD 34+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,
复合物通常对于人类造血发育或特别对于CD 4 + T细胞是必需的。 目标3
将评估HUSH复合物对体内HIV-11潜伏期的贡献。 HUSH复合体的作用
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
个体进行抗HIV-1抑制治疗和来自人源化小鼠。 这些研究预计将
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。
英文摘要
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.
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