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

项目总结 该项目旨在开发和应用CASPEX技术来测量细胞内蛋白质的潜伏期变化 感染了HIV-1病毒。CASPEX是指催化死亡的Cas9(DCas9)蛋白的组合，它可以 定位到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 能够使融合蛋白附近的球体内的蛋白质生物素化。CASPEX支持捕获 指与dCas9复合的引导RNA所定义的基因组位置附近的蛋白质和核酸。 这一强大的方法可以与质谱分析相结合，以识别和量化 以无偏见的方式与基因组基因座相关的蛋白质。 HIV-1潜伏期是开发HIV-1治疗和治愈新疗法的主要障碍。这个 用高通量遗传学研究了控制HIV-1潜伏期的分子过程，小 分子筛选和重点分子生物学研究。CASPEX有潜力提供 关于HIV-1潜伏过程的补充信息，可以确认先前的工作或开辟新途径 研究的成果。在这个项目中，我们将开发两种CASPEX方法，并将它们应用于HIV-1潜伏期的模型。 在具体目标1中，我们将设计HIV-1潜伏期细胞系模型，用于CASPEX分析和表征 潜伏期逆转后，整合的HIV-1染色质基因座上蛋白质的变化。在细胞系中的发现 HIV-1潜伏期的模型并不总是在体内概括，因此在特定的目标2中，我们将开发一种新的 CASPEX在原代CD4T细胞中的应用探讨这一项目的成功完成将推进 对HIV-1潜伏期和潜伏期逆转的分子理解，潜在地为Next-1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.