Reverse Genetics of Arenaviruses.

描述(由申请人提供)：ArenaVirus作为研究急性和持续性病毒感染的易处理的实验模型系统和临床上重要的人类病原体都值得人们极大的兴趣。几种阿拉伯病毒引起人类出血热(HF)疾病，而原型阿拉伯病毒LCMV是研究病毒免疫学和发病机制领域基本概念的极佳工具。此外，有证据表明，LCMV是一种被忽视的具有临床意义的人类病原体。我们的长期目标是对ArenaVirus分子和细胞生物学有一个详细的了解。这些知识将有助于阐明阿拉伯病毒与宿主的相互作用和相关疾病，并有助于制定有效的战略来抗击阿拉伯病毒感染。为此，我们开发了LCMV的反向遗传学系统，为我们提供了一种新的、强大的病毒生物学研究方法。这一建议的重点是对病毒和细胞蛋白质及其相互作用进行功能表征，这些蛋白质和它们之间的相互作用介导了对ArenaVirus RNA合成、颗粒形成和病毒萌发的控制。我们的具体目标是：1.评估Z-NP在控制病毒RNA合成中的作用。我们将检验Z-NP相互作用是Z对病毒RNA合成的抑制活性的假设。我们将使用生化和遗传方法来确定Z-NP相互作用所需的Z和NP区域，并使用反向遗传方法来研究破坏Z-NP相互作用的功能后果。2.确定Z-gp相互作用在病毒感染性颗粒产生中的作用。我们已经证明了感染性VLP的产生需要Z和GP，并证明了Z和GP相互作用。我们将从生化和功能上定义这种Z-gp相互作用。我们将检验这样的假设，即正确的Z-GPcx结合是产生感染性颗粒所必需的，并且这种相互作用是由Z内的特定区域和GP复合体的GP-2成分(GPcx)介导的。3.确定了Z-L结构域在禽流感病毒感染自然过程中的作用。我们将利用反向遗传学来拯救携带不同类型L结构域的Z蛋白的重组人巨细胞病毒。这些重组人巨细胞病毒将用于细胞培养和小鼠感染模型，以检验Z区存在的L结构域类型影响病毒生长和毒力的假设。4.鉴定影响Z-介导萌发的寄主蛋白并对其进行功能鉴定。我们将检验这样一种假设，即含有不同L结构域的Z蛋白与MVB途径中E类蛋白的不同亚群相互作用，这些差异会影响病毒与宿主的相互作用和相关疾病。我们将使用蛋白质组学的方法来定义Z相互作用组。作为补充，我们将使用基于siRNA的筛选来确定有助于Z介导的萌发的细胞因子。我们将使用生化、遗传学和功能分析来评估最初通过蛋白质组和遗传学方法确定的候选病毒在阿拉伯病毒生物学中的相关性。公共卫生相关性： 多种阿拉伯病毒引起人类出血热(HF)疾病，越来越多的证据表明，全球分布的原型性阿拉伯病毒LCMV是一种被忽视的具有临床意义的人类病原体。此外，武器化形式的阿拉伯病毒作为生物恐怖主义的媒介构成严重威胁。目前还没有获得许可的抗ArenaVirus疫苗，目前的抗ArenaVirus治疗仅限于使用利巴韦林，利巴韦林只有部分疗效，而且往往与严重副作用有关。因此，开发新的抗病毒策略来对抗ArenaVirus感染非常重要。对ArenaVirus分子的详细了解将有助于这项任务和细胞生物学，这是本申请中提出的研究的长期目标。

DESCRIPTION (provided by applicant): Arenaviruses merit significant interest both as tractable experimental model systems to study acute and persistent viral infections and as clinically important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans, whereas the prototypic Arenavirus LCMV is a superb workhorse for the investigation of basic concepts in the fields of viral immunology and pathogenesis. In addition, evidence indicates that LCMV is a neglected human pathogen of clinical significance. Our long- term objective is to obtain a detailed understanding of the Arenavirus molecular and cell biology. This knowledge will contribute to the elucidation of Arenavirus-host interactions and associated diseases, and facilitate the development of effective strategies to combat Arenavirus infections. To this end we have developed a reverse genetics system for LCMV that provides us with a novel and powerful approach for the investigation of Arenavirus biology. The focus of this proposal is to functionally characterize viral and cellular proteins, and their interactions, which mediate control of Arenavirus RNA synthesis, particle formation and viral budding. Our specific aims are: 1. To Assess the role of Z-NP In control of viral RNA synthesis. We will test the hypothesis that Z- NP interaction is responsible for the inhibitory activity of Z on viral RNA synthesis. We will use biochemical and genetic approaches to identify the regions of Z and NP required for Z-NP interaction, and use reverse genetic approaches to examine the functional consequences of disrupting Z-NP interaction. 2. Determine the role of Z-GP interaction in production of arenavirus infectious particles. We have demonstrated that production of infectious VLP requires Z and GP, and shown that Z and GP interact. We will define biochemically and functionally this Z-GP interaction. We will test the hypothesis that the correct Z-GPcx association is required for the production of infectious particles, and that this interaction is mediated by defined regions within Z and the GP-2 component of the GP complex (GPcx). 3. Determined the function of Z L domain motifs during the natural course of Arenavirus infection. We will use reverse genetics to rescue rLCMV carrying Z proteins with different types of L-domains. These rLCMV will be used in cell culture and mice models of infection to test the hypothesis that the type of L- domain motifs present in Z influences viral growth and virulence. 4. Identify and functionally characterize host proteins that influence Z-mediated budding. We will test the hypothesis that Z proteins containing different L-domains engage distinct subsets of Class E proteins of the MVB pathway, and that these differences influence virus-host interactions and associated diseases. We will use proteomic approaches to define the Z interactome. As a complementary approach we will use siRNA-based screenings to identify cellular factors that contribute to Z-mediated budding. We will use biochemical, genetics and functional assays to assess the relevance in Arenavirus biology of candidates initially identified by proteomic and genetic approaches. PUBLIC HEALTH RELEVANCE: Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and mounting evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Therefore it is important to develop novel antiviral strategies to combat arenaviral infections. A detailed understanding of the arenavirus molecular will facilitate this task and cell biology, which is the long-term goal of the studies proposed in this application.