Genome-wide shRNA screen to identify cellular regulators of the maintenance of HIV-1 latency

Latent HIV-1 infection represents a barrier to virus eradication as latent HIV-1 is impervious to the effects of antiretroviral drugs and can avoid detection by the host immune system. Strategies to clear latent HIV-1 infection in patients have so far failed in clinical trials to increase the decay rate of the latent reservoir underscoring the need for continued study of HIV-1 latency. In this study, a genome-wide RNAi screen was performed to probe cellular factors involved in maintaining HIV-1 latency in HeLa cells latently infected with an HIV-1 reporter virus. HeLa cells that were latently infected with an HIV-1 reporter virus (referred to as the experimental sample) and uninfected, parental HeLa cells (referred to as the reference control sample) were transduced with the GeneNet Genome-wide Human 50K Lentiviral shRNA Library (System Biosciences (SBI), Mountain View, CA). The library consists of approximately 200,000 shRNA constructs targeting about 38,000 human transcripts. It includes 1-4 shRNA constructs per gene, each targeting a different sequence within a specific gene. Transduced cells were selected with puromycin and cultured for a total of 17 days, which was experimentally determined to provide ample time for shRNA expression and knockdown of the target gene, as well as viral reactivation and viral protein-mediated cell death in the experimental sample. In other words, a negative selection protocol was used to identify shRNAs that activated latent virus by taking advantage of the cytotoxic effects of the HIV-1 viral protein Vpr. Following the culture period, cells were lysed and total RNA was isolated, reverse transcribed, amplified, and hybridized to an Affymetrix GeneChip Array (HG-U133+ 2.0) for identification. As the functional assay employed in this screen involved viral protein-mediated cell death upon the re-activation of latent HIV-1, signals from the reference control sample microarray that were at least three times greater than the corresponding signals from the experimental sample microarray were scored as “hits” and statistically analyzed further.

潜伏性HIV-1感染是病毒根除的一大障碍，因为处于潜伏状态的HIV-1不受抗逆转录病毒药物的作用影响，且能够逃逸宿主免疫系统的检测。迄今为止，旨在清除患者体内潜伏性HIV-1感染的各类策略，在临床试验中均未能提高潜伏病毒库的衰减速率，这凸显了持续开展HIV-1潜伏机制研究的必要性。本研究通过全基因组RNA干扰（RNA interference, RNAi）筛选，探究在携带HIV-1报告病毒的潜伏感染HeLa细胞中，参与维持HIV-1潜伏状态的宿主细胞因子。本研究将GeneNet全人类基因组50K慢病毒短发夹RNA（short hairpin RNA, shRNA）文库（系统生物科学公司（SBI），美国加利福尼亚州山景城）转导至两组细胞：一组为携带HIV-1报告病毒的潜伏感染HeLa细胞（记为实验组样本），另一组为未感染的亲本HeLa细胞（记为参考对照样本）。该文库包含约20万个shRNA构建体，可靶向约38000个人类转录本。每个基因对应1至4个shRNA构建体，每个构建体靶向特定基因内的不同序列。转导后的细胞用嘌呤霉素进行筛选，总培养时长为17天；该时长经实验确定，可使shRNA充分表达并完成靶基因的敲低，同时也为实验组样本中的病毒重新激活以及病毒蛋白介导的细胞死亡提供了充足时间。换言之，本研究采用负筛选策略，利用HIV-1病毒蛋白Vpr的细胞毒性作用，筛选可激活潜伏病毒的shRNA。培养周期结束后，裂解细胞并分离总RNA，经逆转录、扩增后，与Affymetrix基因芯片阵列（HG-U133+ 2.0）进行杂交以完成靶点鉴定。由于本筛选所采用的功能实验涉及潜伏HIV-1重新激活后病毒蛋白介导的细胞死亡，因此，参考对照样本的芯片信号强度若至少为对应实验组样本芯片信号强度的三倍，则被判定为阳性命中靶点，并对其开展进一步的统计学分析。