Characterization of ADAR, a key RNA editing protein with pro-viral activities

说明(申请人提供)：干扰素(IFN)是宿主细胞释放的一大类免疫调节蛋白。I型干扰素因其对病毒感染的保护作用而备受关注，它们还可用于治疗癌症、炎症性肠病和多发性硬化症。I型干扰素与其受体的结合启动了一系列信号级联反应，诱导了数百个干扰素刺激基因(ISGs)的表达，其中大多数还没有完全确定。莱斯实验室之前的一项研究筛选了数百个ISG对各种病毒的抗病毒作用。从这个屏幕上可以看到一个ISG，ADAR1(作用于RNA 1的腺苷脱氨酶)--特别是ADAR1p150--增强而不是抑制一组临床上重要的正链(+)RNA病毒的复制；西尼罗河病毒(WNV)、基孔肯雅病毒(CHIKV)、委内瑞拉马脑炎病毒(VEEV)和辛德比斯病毒(SINV)。ADAR1催化双链RNA(DsRNA)中腺苷(A)脱氨为肌苷(I)，也是一种不依赖于其编辑活性的dsRNA结合蛋白。我们的主要假设是ADAR1p150是A对I编辑病毒或宿主dsRNA，从而导致(+)RNA病毒复制的增强。这项建议的主要目的是了解ADAR1p150增强(+)RNA病毒复制的机制，并进一步确定ADAR1p150在宿主和病毒中的RNA靶标。目的1确定ADAR1p150通过突变ADAR1p150蛋白的不同结构域--包括负责RNA编辑的脱氨酶结构域--来增强病毒复制的机制，以确定增强(+)RNA病毒复制所需的ADAR1p150的结构域/活性。除了定义增强病毒复制所需的关键ADAR1活动外，还将确定受影响的病毒生命周期步骤(S)。AIM 2将通过在表达WT或突变体ADAR1p150的细胞中进行高通量RNA测序，进一步鉴定宿主和病毒中由ADAR1p150编辑或与ADAR1p150结合的RNA序列。目前还没有针对SINV、WNV、CHIKV和VEEV的特定治疗方法。此外，ADAR1是人类免疫缺陷病毒(HIV)的亲病毒ISG，以及丙型肝炎病毒(HCV)的抗病毒ISG，这两种全球流行病导致许多人死亡。因此，努力了解ADAR1的机制和靶点可能会导致新的治疗方法来对抗这些不同的病毒感染。重要的是，ADAR1在小鼠体内的缺失会导致快速的细胞凋亡、造血缺陷和干扰素信号的全球上调，从而导致胚胎死亡。然而，对于ADAR1在哺乳动物宿主中的作用机制及其特异的RNA靶点，我们仍然没有一个清晰的认识。因此，在未感染和病毒感染的细胞中研究ADAR1的机制和靶点的结果将为理解ADAR在宿主生物学中的作用提供宝贵的资源。

DESCRIPTION (provided by applicant): Interferons (IFNs) are a large class of immunomodulatory proteins released by host cells. Type I IFNs attract much attention due to their protective role against viral infections, and they may also be used to treat cancer, inflammatory bowel diseases, and multiple sclerosis. Binding of type I IFNs to its receptor initiates a signaling cascade that induces the expression of hundreds of IFN-stimulated genes (ISGs), the majority of which are not fully characterized. A previous study from the Rice lab screened hundreds of ISGs for antiviral effects on diverse viruses. From this screen one ISG, ADAR1 (adenosine deaminases acting on RNA 1) - specifically the isoform ADAR1p150 - enhanced rather than inhibited replication of a group of clinically important positive- strand (+)RNA viruses; West Nile virus (WNV), chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), and Sindbis virus (SINV). ADAR1 catalyzes deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA), and also functions as a dsRNA binding protein independent of its editing activity. Our primary hypothesis is that ADAR1p150 is A-to-I editing a virus or host dsRNA, which leads to the enhancement of (+)RNA virus replication. The main goal of this proposal is to understand the mechanism by which ADAR1p150 enhances (+)RNA virus replication, and to further identify RNA targets of ADAR1p150 in the host and virus. Aim 1 proposes to determine the mechanism by which ADAR1p150 enhances virus replication by mutating different domains of the ADAR1p150 protein - including its deaminase domain responsible for RNA editing - to determine the domains/activities of ADAR1p150 required to enhance (+)RNA virus replication. In addition to defining the critical ADAR1 activities required for enhanced virus replication, the affected virus lifecycle step(s) will be determined. Aim 2 will further identify RNA sequences 'edited by' or 'bound to' ADAR1p150 in the host and virus by performing high-throughput RNA-sequencing in cells expressing WT or mutant ADAR1p150. Currently there is no specific treatment available for SINV, WNV, CHIKV, and VEEV. Moreover, ADAR1 is a pro-viral ISG for human immunodeficiency virus (HIV), and an antiviral ISG for hepatitis C virus (HCV), two global pandemics that cause numerous human deaths. Thus, efforts to understand the mechanism and targets of ADAR1 may lead to new therapeutics to combat these various viral infections. Importantly, ADAR1 deletion in mice causes rapid apoptosis, defective hematopoiesis, and a global up-regulation in IFN signaling, which lead to embryonic lethality. However, we still do not have a clear picture of ADAR1 mechanism of action and its specific RNA targets in the mammalian host. Therefore, results gained from examining ADAR1 mechanism and targets in uninfected and virally infected cells will serve as a valuable resource to understand ADAR's roles in host biology.