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

说明(申请人提供)：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冠状病毒的出现和禽流感发病率的增加突显了新疫苗和疗法在防止疾病和疾病在世界各地传播的潜在重要性。这项研究的基本原理是，SARS-CoV和所有其他专性细胞内病原体一样，其生命周期需要宿主蛋白，抑制关键宿主蛋白的表达应该会阻止病毒复制。我们已经成功地使用基因陷阱插入突变来随机灭活细胞基因，作为一种工具来快速识别甲型流感、HIV-1、马尔堡、埃博拉或呼肠孤病毒复制所需的新宿主基因，而不是宿主细胞生存所需的新宿主基因。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已经失活。我们发现了几条被无关病毒使用的途径，这表明存在病毒复制的保守机制，并且使用小干扰RNA(SiRNA)抑制这些途径可能允许进行广泛的治疗干预。目前应用的目标是将基因捕获技术扩展到研究SARS-CoV感染的新的宿主遗传要求，并利用RNA干扰作为一种策略来独立确认这些基因在病毒复制中的要求。我们的具体目标是：1)使用Vero E6细胞基因捕捉库来筛选抗SARS-CoV感染的细胞，并确定其干扰赋予耐药表型的候选基因；2)表征在特定目标1中确定的沉默候选基因对SARS-CoV复制、宿主细胞活力和生长速度的影响。这项研究旨在确定在不对宿主细胞造成有害影响的情况下抑制SARS-CoV复制的特定siRNA。对公众健康的好处是，瞬时击倒宿主基因mRNAs以阻断感染性病原体可以提供广谱治疗干预，而通过病原体的突变即使不是不可能，也很难绕过这一点。这项应用的目标是确定SARS-CoV复制所必需的新的宿主基因要求。在SARS-CoV复制中起关键作用的候选宿主基因将通过随机突变宿主基因，然后在存活的SARS-CoV抗性克隆细胞系中识别中断基因来识别。RNA干扰(RNAi)研究将证实SARS-CoV复制过程中对候选基因的要求。在RNAi筛选之后，将产生针对关键基因的短发夹状(ShRNA)表达细胞系，以确定长期靶基因沉默对细胞生长速度和活力的影响，并为超出该应用期限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.