HSV-1诱导宿主miR-433的功能与机制研究

单纯疱疹病毒Ⅰ型(HSV-1)能在外周神经系统(PNS)终生潜伏，其周期性重激活引发疱疹和角膜炎，甚至病毒性脑炎，但CD4+、CD8+T细胞不能清除被感染细胞。我们用ChIP-seq和RNA-seq发现病毒感染后宿主miR433转录增加。miR433抑制NK细胞表面受体NKG2D的配体MICB表达，病毒感染的细胞上调MICB而被NK细胞杀伤，我们推测HSV-1诱导宿主miR-433是为了逃避该反应。为验证假说，我们计划筛选诱导miR-433转录的病毒因子并找到miR-433的顺式作用元件，揭示HSV-1激活miR433转录的机制；用HSV-1感染及转染病毒蛋白等方式验证病毒免疫逃避机制；制备无法激活miR-433转录的缺陷病毒，并在小鼠和树鼩模型上检测潜伏和重激活能力的变化。这个项目将揭示HSV-1，甚至疱疹病毒属潜伏感染和重激活的一个关键机制，并为临床干预和治疗疱疹病毒性疾病提供新靶点。

Following primary infection, Herpes Simplex Virus type I (HSV-1) establishes life long latent infections in peripheral nervous system (PNS) sensory neurons, and its reactivation leads to recurring cold sores, genital Herpes, Herpes Simplex Keratitis and, in rare but often fatal cases, Herpes Simplex Encephalitis. In latent infected human Trigeminal Ganglia, CD4+CD8+ lymphocytes are detected, but they were unable to clear the infected neurons, indicating effective evasion from host immune surveillance. To determine the mechanism by which HSV-1 employs to hide from host immunity, we did ChIP-seq and RNA-seq analyses of HSV-1 infected transcriptome and epigenome, and discovered an increase of RNA Pol II binding to, and elevated expression of host miR433. miR433 is one of the cellular micro RNA targeting the MHC I class molecule MICB (MHC class I related molecule B), an activating ligand for Natural Killer (NK) cell surface receptor NKG2D. Its purpose is to attenuate the level of MICB, so that normal cells would not be accidentally targeted by NK cells. In viral infected cells, cancer cells and cells with extensive DNA damage, MICB level goes up, making these cells visible to NK cells for elimination. We hypothesize that HSV-1 up regulates miR433 to counteract this process in order to hide from host native immunity. To test his hypothesis, we plan to 1) determine the mechanism by which HSV-1 activates miR433 through the identification of viral factors responsible for miR433 activation, and determine the cis regulatory element responsible for miR433 activation by HSV-1 infection via ChIP-seq analyses of key chromatin marks associated with gene enhancers such as H3K27AC, H3K4me3; 2) using HSV-1 infection and transfected viral factors to functionally confirm that the up regulation indeed lead to reduced recognition by NK cells; 3) Making mutant virus lacking the ability to induce miR433, and to study the effect of the mutation in mouse and tree shrew latency models to test whether the ability to establish latency or the ability to reactivate are affected in the mutant virus. This study, if funded, would reveal a key mechanism on how HSV-1 and other viruses in the Herpes family successfully establish latent infections and mount recurring reactivation, and provided important new therapeutic targets for clinical intervention.