Ex Vivo Detection and Analysis of Non-inducible Latent HIV

 描述（由申请人提供）：目前没有方法能够有效地“休克”细胞，使其从潜伏的HIV储库中产生病毒，这是免疫系统杀死抗原阳性细胞的先决条件。了解阻碍前病毒诱导出潜伏期的机制对潜伏期逆转和水库枯竭至关重要。我们建议 通过开发和实施定义潜伏感染细胞特性的新方法来识别这些机制中的至少一些。在拟议研究的R21阶段，我们将开发一个实验管道，该管道将能够分离和> 1，000倍扩增携带单个诱导型或非诱导型前病毒的细胞，纯度至少为 1%（相比之下，未分级PBMC群体中约为0.001%）。然后，我们将通过多位点qPCR筛选确定这些前病毒的结构，然后进行完整的前病毒测序，以确定编码感染性病毒的潜力。该程序还将揭示宿主染色体中的前病毒整合位点和附加型环状病毒DNA的存在。在R33阶段，我们将使用病毒生长测定区分诱导型和非诱导型前病毒，并将尝试使用扩增的细胞群使用PCR激活的细胞分选分离纯的潜伏感染细胞。此外，我们将通过对上述扩增的细胞谱系和含有诱导型和非诱导型前病毒的纯化细胞群进行多重同时下游分析，进一步开发我们的储库表征新方法。这些将包括前病毒和邻近细胞基因的转录活性、病毒蛋白质产生和DNA甲基化模式。这些研究可能揭示防止病毒诱导潜伏的机制。此外，通过扩增和鉴定携带单个诱导型和非诱导型前病毒的细胞群，我们还将为诱导前病毒脱离潜伏期的离体测试手段提供有价值的资源。

 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.