Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, Washington, United States of America.
Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.
PLoS Pathog. 2023 Jan 27;19(1):e1011101. doi: 10.1371/journal.ppat.1011101. eCollection 2023 Jan.
Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.
潜伏 HIV-1 前病毒的转录沉默需要复杂且重叠的机制，这是体内消除 HIV-1 的主要障碍。我们开发了一种新的潜伏 CRISPR 筛选策略，称为 Latency HIV-CRISPR，它使用包装有指导 RNA 编码慢病毒载体基因组的出芽病毒上清液作为直接读出物，用于检测参与 HIV-1 潜伏维持的因素。我们开发了一个针对表观遗传调节基因的定制指导 RNA 文库，并将该筛选与潜伏逆转剂 AZD5582（非经典 NFκB 途径的激活剂）配对，以检查控制 HIV-1 潜伏的多种机制。组蛋白 H4 乙酰转移酶的核小体乙酰转移酶（NuA4 HAT）复合物的一个组成部分，ING3，与 AZD5582 协同作用，激活 J-Lat 细胞系和 HIV-1 潜伏的原代 CD4+T 细胞模型中的前病毒。我们发现，ING3 的敲除会降低 H4 组蛋白尾部的乙酰化和 BRD4 在 HIV-1 LTR 上的占据。然而，ING3 敲除与 AZD5582 激活非经典 NFκB 途径的组合导致 HIV-1 前病毒上 RNA 聚合酶 II 的起始和延伸急剧增加，这种方式在所有细胞启动子中几乎是独一无二的。
