Characterizing chromatin protein dynamics in HIV-1 latency with a CASPEX approach

项目概要 该项目旨在开发和应用CASPEX技术来测量细胞中潜在的蛋白质变化 感染 HIV-1。 CASPEX 是指催化死亡 Cas9 (dCas9) 蛋白的组合，可以 定位于与工程抗坏血酸过氧化物酶 (APEX2) 融合的特定基因组位点，该酶是 能够对融合蛋白附近范围内的蛋白质进行生物素化。 CASPEX 启用捕获 靠近由与 dCas9 复合的指导 RNA 定义的基因组位点的蛋白质和核酸。 这种强大的方法可以与质谱分析相结合，以识别和量化 以公正的方式与基因组位点相关的蛋白质。 HIV-1 潜伏期是开发 HIV-1 治疗和治愈新疗法的主要障碍。的 控制 HIV-1 潜伏期的分子过程已经使用高通量遗传学、小 分子筛选和重点分子生物学研究。 CASPEX 有潜力提供 有关 HIV-1 潜伏过程的补充信息，可以确认之前的工作或开辟新的途径 研究。在这个项目中，我们将开发两种 CASPEX 方法并将其应用于 HIV-1 潜伏期模型。 在具体目标 1 中，我们将设计 HIV-1 潜伏期的细胞系模型，用于 CASPEX 分析并表征 HIV-1 染色质整合位点的蛋白质变化响应潜伏期逆转。细胞系中的发现 HIV-1 潜伏期模型并不总是在体内重现，因此在特定目标 2 中，我们将开发一种新的 在原代 CD4 T 细胞中应用 CASPEX 的方法。该项目的顺利完成将推动 对 HIV-1 潜伏期和潜伏期逆转的分子理解，可能为下一代的发展提供信息 治疗或治愈 HIV-1 感染的新一代疗法。

PROJECT SUMMARY This project aims to develop and apply CASPEX technologies to measure protein changes in cells latently infected with HIV-1. CASPEX refers to the combination of a catalytically-dead Cas9 (dCas9) protein that can localize to a specific genomic locus fused to an engineered ascorbate peroxidase enzyme (APEX2) that is capable of biotinylating proteins within a sphere of proximity to the fusion protein. CASPEX enables the capture of proteins and nucleic acids in proximity to a genomic locus defined by guide RNAs complexed with dCas9. This powerful approach can be combined with mass spectrometry analysis to identify and quantify changes in proteins associated with genomic loci in an unbiased manner. HIV-1 latency represents the major barrier to developing new therapies for HIV-1 treatment and cure. The molecular processes governing HIV-1 latency have been investigated using high-throughput genetics, small molecule screening, and focused molecular biology studies. CASPEX has the potential to provide complementary information regarding HIV-1 latency processes that can confirm prior work or open new avenues of research. In this project, we will develop two CASPEX approaches and apply them to models of HIV-1 latency. In Specific Aim 1, we will engineer cell line models of HIV-1 latency for CASPEX analysis and characterize protein changes at integrated HIV-1 chromatin loci in response to latency reversal. Findings made in cell line models of HIV-1 latency are not always recapitulated in vivo, so in Specific Aim 2 we will develop a novel approach to apply CASPEX in primary CD4+ T cells. Successful completion of this project will advance the molecular understanding of HIV-1 latency and latency reversal, potentially informing on the development of next- generation therapies to treat or cure HIV-1 infection.