RNAi as an Intercellular Antiviral Defense Mechanism

描述(申请人提供)：RNA干扰(RNAi)是双链RNA(DsRNA)引导的机制，介导RNA的序列特异性降解。最近证明了RNA干扰可以用来抑制病毒复制，这开启了一个令人兴奋的研究领域。首先，它提供了一种新的抗病毒治疗方法，其次，它可能构成一种迄今未被认识的天然抗病毒防御机制。这项建议的长期目标是更好地了解RNA干扰(RNAi)在抑制病毒感染方面的意义和治疗潜力。为此，我们提出了三个具体目标：(1)了解基于siRNA的病毒抑制的决定因素；(2)研究RNAi作为一种天然的抗病毒防御机制；以及(3)识别和表征RNAi机制的新组件。我们将使用特性良好的正链RNA病毒(小核糖核酸病毒、甲型病毒和黄病毒)作为模型病毒，以果蝇和哺乳动物细胞作为模型宿主。(1)为了了解基于siRNA的病毒抑制的决定因素，我们将定义对RNAi敏感的RNA病毒的基因组区域，并探索不同siRNA设计方法的效率。我们还将调查病毒逃避RNAi的能力。(2)为了研究RNAi作为一种天然的抗病毒防御机制，我们检测了病毒感染动物细胞是否产生siRNAs，并分析了RNAi机制的抑制成分是否促进了病毒在哺乳动物细胞中的复制。我们还将确定病毒感染是否会导致RNAi机制的变化。(3)为了鉴定和表征RNAi机制的新组件，我们在果蝇细胞中进行了全基因组筛选。然后，我们将使用细胞生物学和生物化学来表征进入机制和在我们的筛选中发现的基因的作用，并分离在我们的筛选中发现的RNAi进入成分的哺乳动物同源基因，并探索它们在哺乳动物系统中的RNAi进入中的作用。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is a double-stranded RNA (dsRNA) guided mechanism that mediates sequence specific degradation of RNA. The recent demonstration that RNA interference can be used to inhibit virus replication has initiated an exciting field of research. First, it provides a novel antiviral therapeutic approach and, second, it may constitute a hitherto unrecognized natural antiviral defense mechanism. The long-term goal of this proposal is to gain a better understanding of the significance and therapeutic potential of RNA interference (RNAi) in the inhibition of viral infections. To this end we propose three specific aims to: (1) understand the determinants of siRNA-based viral inhibition; (2) investigate RNAi as a natural antiviral defense mechanism; and (3) identify and characterize novel components of the RNAi machinery. We will employ well-characterized positive stranded RNA viruses (picornaviruses, alphaviruses, and flaviviruses) as model viruses, and employ Drosophila and mammalian cells as model hosts. (1) To understand the determinants of siRNA-based viral inhibition, we will define the genome regions of RNA viruses that are susceptible to RNAi, and explore the efficiency of different siRNA design approaches. We will also investigate the ability of viruses to escape RNAi. (2) To investigate RNAi as a natural antiviral defense mechanism, we examine whether viral infection of animal cells produces siRNAs and analyze whether inhibiting components of the RNAi machinery enhances viral replication in mammalian cells. We will also determine whether viral infection induces changes in the RNAi machinery. (3) To identify and characterize novel components of the RNAi machinery, we carry out genome-wide screens in Drosophila cells. We will then employ cell biology and biochemistry to characterize the mechanism of entry and the role of the genes identified in our screens, and isolate the mammalian orthologs of RNAi entry components identified in our screens and explore their role in RNAi entry in mammalian systems.