Ex Vivo Detection and Analysis of Non-inducible Latent HIV

 描述(由申请人提供)：目前没有一种方法能够有效地将细胞从潜伏的艾滋病毒储存库“休克”成病毒，这是免疫系统杀死抗原阳性细胞的先决条件。了解阻碍诱导潜伏期前收缩的机制对于潜伏期逆转和储备库耗尽至关重要。我们建议 通过开发和实施一种新的方法来定义潜伏感染细胞的属性，至少确定其中一些机制。在拟议研究的R21阶段，我们将开发一种实验管道，该管道将能够分离和1000倍扩增含有单个可诱导或非诱导前病毒的细胞，纯度至少为 1%(与之相比，在未分离的PBMC群体中约为0.001%)。然后，我们将通过多位点qPCR筛查和完整的前病毒测序来确定这些前病毒的结构，以确定编码传染性病毒的可能性。这一过程还将揭示宿主染色体中的前病毒整合位置和存在的上体、环状病毒DNA。在R33阶段，我们将使用病毒生长试验区分可诱导和不可诱导的前病毒，并尝试使用扩增的细胞群来分离纯的、潜伏感染的细胞，使用聚合酶链式反应激活的细胞分类。此外，我们将进一步发展我们的新方法，通过对上述扩展细胞谱系和含有可诱导和非诱导前病毒的纯化细胞群体进行多个同步下游分析，来确定储集层的特征。这些将包括前病毒和邻近细胞基因的转录活性、病毒蛋白生产和DNA甲基化模式。这些研究可能揭示阻止潜伏期病毒诱导的机制。此外，通过扩大和鉴定含有单个可诱导和不可诱导前病毒的细胞群，我们还将为体外诱导潜伏期前病毒的方法提供宝贵的资源。

 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.