Determining SARS-CoV host gene requirements by mutagenesis and RNA interface

描述（由申请方提供）：呼吸道病毒感染是发达国家和欠发达国家发病率、死亡率和经济损失的主要来源。SARS-CoV的出现和禽流感发病率的增加突出了新疫苗和治疗方法在预防疾病和疾病在世界各地传播方面的潜在重要性。这项研究的基本原理是，SARS冠状病毒，像所有其他专性细胞内病原体，需要宿主蛋白质的生命周期，抑制关键宿主蛋白质的表达应该阻止病毒复制。我们已经成功地使用基因陷阱插入诱变随机扩增细胞基因，作为快速鉴定甲型流感、HIV-1、马尔堡、埃博拉或呼肠孤病毒复制所需的新宿主基因的工具，但不是宿主细胞存活所需的。病毒感染通常会杀死选定的宿主细胞系，除非病毒生命周期所必需的基因被灭活。我们确定了几种不相关的病毒使用的途径，这表明存在保守的病毒复制机制，并且使用小干扰RNA（siRNA）抑制这些途径可能允许广谱治疗干预。本申请的目的是将基因捕获技术扩展到研究SARS-CoV感染的新宿主遗传要求，并使用RNA干扰作为独立确认病毒复制中这些基因的要求的策略。我们的具体目标是：1）使用Vero E6细胞基因捕获文库来选择对SARS-CoV感染具有抗性的细胞，并鉴定其破坏赋予抗性表型的候选基因，和2）表征在具体目标1中鉴定的沉默候选基因对SARS-CoV复制和宿主细胞活力和生长速率的影响。本研究旨在鉴定特异性siRNA，其抑制SARS-CoV复制而不对宿主细胞造成有害影响。对公共健康的益处是宿主基因mRNA的瞬时敲低以阻断感染性病原体可以赋予广谱治疗干预，这是难以（如果不是不可能的话）通过病原体突变来规避的。本申请的目的是确定SARS冠状病毒复制所必需的新宿主遗传要求。通过随机突变宿主基因，然后在存活的克隆SARS-CoV抗性细胞系中鉴定被破坏的基因，来鉴定在SARS-CoV复制中起关键作用的候选宿主基因。SARS冠状病毒复制对候选基因的需求将在RNA干扰（RNAi）研究中得到证实。在RNAi筛选之后，将产生表达针对关键基因的短发夹（shRNA）的细胞系，以确定长期靶基因沉默对细胞生长速率和活力的影响，并为本申请期限之外的前瞻性机制研究做准备。

DESCRIPTION (provided by applicant): Respiratory virus infections are a major source of morbidity, mortality, and economic loss in both developed and under-developed countries. The emergence of SARS-CoV and the increased incidence of avian influenza highlight the potential importance of new vaccines and therapeutics to prevent disease and disease spread around the world. The rationale for this study is that SARS-CoV, like all other obligate intracellular pathogens, requires host proteins for its life cycle and that inhibiting the expression of critical host proteins should block viral replication. We have successfully used gene trap insertional mutagenesis to randomly inactivate cellular genes as a tool for rapidly identifying novel host genes required for influenza A, HIV-1, Marburg, Ebola, or reovirus replication, but not host cell survival. Viral infection normally kills the chosen host cell line unless a gene essential for the viral life cycle has been inactivated. We identified several pathways used by unrelated viruses suggesting the existence of conserved mechanisms for viral replication, and that inhibiting such pathways using small interfering RNA (siRNA) may allow broad spectrum therapeutic intervention. The objectives of the current application are to extend gene trap technology towards the study of novel host genetic requirements for SARS-CoV infection, and to use RNA interference as a strategy to independently confirm the requirement of these genes in viral replication. Our Specific Aims are to 1) use Vero E6 cell gene trap libraries to select cells resistant to SARS- CoV infection and identify candidate genes whose disruption confer the resistant phenotype, and 2) to characterize the effects of silencing candidate genes identified in Specific Aim 1 on SARS-CoV replication and host cell viability and growth rates. This study is designed to identify specific siRNAs that inhibit SARS-CoV replication without causing detrimental effects to host cells. The benefit to public health is that the transient knock- down of host gene mRNAs to block infectious pathogens could confer broad spectrum therapeutic intervention that is difficult, if not impossible, to circumvent through mutation of the pathogen. The goal of this application is to determine novel host genetic requirements essential for SARS-CoV replication. Candidate host genes performing critical roles in SARS-CoV replication will be identified by randomly mutating host genes, and then identifying disrupted genes in surviving clonal SARS-CoV-resistant cell lines. The requirement of candidate genes in SARS-CoV replication will be confirmed in RNA interference (RNAi) studies. Following RNAi screening, cell lines expressing short hairpin (shRNA) against critical genes will be generated to determine the effects of long term target gene silencing on cellular growth rates and viability, and to prepare for prospective mechanistic studies beyond the term of this application.