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Ex Vivo Detection and Analysis of Non-inducible Latent HIV

非诱导性潜伏 HIV 的离体检测和分析

基本信息

批准号：
9046237
负责人：
JAMES Ivan MULLINS
金额：
$ 23.64万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9046237
关键词：
Abate Address Antigens Biological Assay CD4 Positive T Lymphocytes Cell Count Cell Density Cell Fraction Cell Lineage Cell Proliferation Cell Separation Cells Chromosomes Consensus Cytolysis DNA DNA Methylation Detection Development Environment Frequencies Gene Expression Genes Genetic Transcription Genome Gold HIV HIV Infections Immune system In Vitro Individual Interleukin-2 Laboratories Latent Virus Metabolism Methodology Methods Peripheral Blood Mononuclear Cell Phase Population Procedures Production Property Protocols documentation Proviruses RNA Reading Regimen Residual state Resources Rest Sampling Shock Signal Transduction Structure T-Lymphocyte Technology Testing Time Transcript Viral Viral Proteins Virus Virus Activation Virus Integration Virus Latency bisulfite sequencing in vivo integration site killings methylation pattern novel novel strategies prevent protein expression public health relevance single cell analysis standard measure viral DNA whole genome

项目摘要

DESCRIPTION (provided by applicant): No current methods are able to efficiently "shock" cells into producing virus from latent HIV reservoirs, a prerequisite for killing antigen positive cells by the immune system. Understanding the mechanisms that hinder the induction of proviruses out of latency is critical to latency reversal and to reservoir depletion. We propose to identify at least some of these mechanisms through development and implementation of a new approach to defining properties of latently infected cells. In the R21 phase of the proposed studies we will develop an experimental pipeline that will enable isolation and >1,000-fold expansion of cells harboring a single inducible or a non-inducible provirus at a purity of at least 1% (compared to ~0.001% in unfractionated PBMC populations). We will then define the structure of these proviruses by a multilocus qPCR screen followed by complete provirus sequencing to determine the potential for encoding infectious virus. This procedure will also reveal the proviral integration sites in host chromosomes and presence of episomal, circular viral DNA. In the R33 phase, we will distinguish between inducible and non-inducible proviruses using virus outgrowth assays and will attempt to use the expanded cell populations for isolation of pure, latently infected cells using PCR-activated cell sorting. In addition, we will further develop our new approach to reservoir characterization by performing multiple simultaneous downstream analyses of the aforementioned expanded cell lineages and purified cell populations harboring inducible and non-inducible proviruses. These will include transcriptional activity of the provirus and adjacent cellular genes, viral protein production and DNA methylation patterns. These studies are likely to reveal mechanisms preventing viral induction from latency. In addition, by expanding and identifying cell populations harboring individual inducible and non-inducible proviruses we will also provide a valuable resource for ex vivo testing means of inducing proviruses out of latency.