Identification of HIV latency biomarkers with a dual fluorescence reporter HIV

 描述(由申请人提供)： 摘要病毒潜伏期已成为根除HIV-1感染的主要障碍。目前消除病毒库的理论范式被称为“休克和杀死”--即使用非靶向小分子重新激活潜伏病毒。然而，要使这一方法取得成功，必须克服重大障碍。这些措施包括随机病毒重新激活，避免全球T细胞激活，以及病毒重新激活时有限的细胞死亡。因此，针对HIV-1潜伏期的另一种策略将利用生物标记物来识别和选择性地靶向体内潜伏感染的细胞，而不需要重新激活。此外，这些生物标记物对于体外研究将是无价的，因为现在可以提纯患者来源的、自然状态潜伏感染的细胞进行研究。然而，不幸的是，目前还不存在这样的生物标志物。在这里，我们提出了一种多管齐下的、基于系统生物学的策略来识别原始CD4+T细胞中的潜伏期生物标记物。我们建议使用一种新的双荧光报告HIV-1基因组来识别、量化和纯化感染后早期(&lt；4天)的潜伏感染细胞，而不需要重新激活。用这种病毒感染原代CD4+T细胞会在约30%的感染事件中产生可逆的HIV潜伏期状态， 是一个比当前使用的许多其他主小区延迟模型高得多的频率。该模型的一组独特的特征是我们建议使用强大的表型工具组合来表征在HIV-1感染初级CD4+T细胞后变得潜伏的细胞的基础。这些工具将包括：1。细胞表面标志物、T细胞信号网络和T细胞效应器功能的单细胞分析。2.基于定量质谱学的鉴定在潜伏感染细胞和高产感染细胞上富含的细胞表面蛋白；3.比较潜伏感染细胞和高产感染细胞的细胞转录组的RNAseq和microRNAseq分析。最重要的是，我们还将筛选我们的假定生物标记物清单，以了解它们的预测作用。这不仅将在体外感染双荧光病毒的细胞中进行验证，而且将在体外通过从HIV感染患者分离的CD4+T细胞中进行验证。我们认为，这种多管齐下的验证方法对于识别真正的生物标记物至关重要，并可能产生对新型HIV-1潜伏期调节疗法的体外验证非常有价值的靶点。

 DESCRIPTION (provided by applicant): ABSTRACT Viral latency has emerged as the main barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill'-i.e. reactivation of latent virus using non- targeted small molecules. However, significant hurdles must be overcome for this approach to be successful. These include stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upon viral reactivation. Thus, an alternative strategy for targeting HIV-1 latency would make use of biomarkers to identify and selectively target latently infected cells in vivo without the need for reactivation. Additionally, these biomarkers would be invaluable for in vitro research given that patient-derived, native-state latently infected cells could now be purified for study. Unfortunatel however, such biomarkers do not currently exist. Here, we propose a multi-pronged, systems-biology based strategy to identify latency biomarkers in primary CD4+ T-cells. We propose to use a novel dual-fluorescence reporter HIV-1 genome to identify, quantify, and purify latently infected cells early post infection (<4 days), and without reactivation. Infection of primary CD4+ T-cells with this virus yields a reversible state of HIV latency in ~30% of infection events, which is a much higher frequency than many other currently used primary cell latency models. This model's unique set of characteristics is fundamental to our proposal to use a powerful combination of phenotyping tools to characterize the cells that become latent after HIV-1 infection of primary CD4+ T-cells. These tools will include: 1. CyTOF single-cell analysis of cell-surface markers, T-cell signaling networks, and T-cell effector function. 2. Quantitative mass spectrometry based identification of cell surface proteins enriched on latently infected cells in comparison to productively infected cells; 3. RNAseq and microRNAseq analysis of the cellular transcriptome of latently infected cells in comparison to productively infected cells. Most importantly, we will also screen our list of putative biomarkers for their predictive utility. Thiswill be done by validation not only in cells infected in vitro with the dual fluorescence virus, but als ex vivo in CD4+ T-cells isolated from HIV infected patients. We believe that this multi-pronged validation approach is vital to identifying bona fide biomarkers, and is likely to yield targets tht are invaluable for ex vivo validation of novel HIV-1 latency modulating therapeutics.