Viral and Host Determinants of New World Alphaviral Entry

描述（由申请人提供）：东部马脑炎病毒（EEEV）是人类致病性最高的病毒之一，死亡率为35-50%，是一种重新出现的公共卫生威胁。EEEV是一种新世界甲病毒，是一组蚊媒脑病病原体，具有广泛的地理分布和宿主范围，包括相关媒介西方马脑炎病毒（WEEV）和委内瑞拉马脑炎病毒（VEEV）。更好地了解新世界甲型病毒进入宿主细胞的病毒和宿主决定因素将有助于深入了解病毒生命周期和致病性的关键方面，并可能为未来治疗策略和疗法的发展提供基础。由于对特殊生物安全控制设施的要求，对新世界甲型病毒的趋向性和进入性的分子认识受到限制。为了克服这些限制，我们开发了“假型”病毒，其表面含有EEEV， WEEV或VEEV包膜糖蛋白，并表达作为感染标记的报告基因。每个假型病毒都概括了它们所基于的特定病毒的原生进入机制，并且可以在标准的BSL-2条件下使用。我们还生成了可溶的新世界α病毒E2糖蛋白外结构域，可以结合允许的靶细胞。这些试剂能够对具有代表性和重要的新世界甲病毒的病毒进入途径的分子细节进行比较研究。本提案的目的是确定新世界甲病毒包膜糖蛋白内的受体结合域，确定新世界甲病毒是否具有共同的细胞表面受体，并确定其特异性宿主进入因子。在第一个目标中，候选人将产生可溶形式的新世界α病毒E2糖蛋白，具有迭代的N和c端截断和位点定向突变，并表征其结合靶细胞的能力。在第二个目标中，候选人将比较表征新世界α病毒在广泛靶细胞上的趋向性，通过一系列结合和感染竞争绘制常见受体的使用情况，并通过可溶性α病毒E2受体结合域的免疫沉淀识别特定的细胞表面受体。在Broad研究所的支持下，在第三个目标中，候选人将通过使用shRNA文库对不允许新世界甲型病毒伪型感染的人类细胞进行高通量遗传筛选，确定特定的宿主细胞进入因子。在新英格兰生物防御和新发传染病卓越区域中心（NERCE BEID） BSL-3核心设施的活病毒实验中，用可溶性受体结合域鉴定的新世界甲型病毒或使用假型病毒的遗传筛选的潜在宿主因子的重要性将得到证实。这些研究将在Michael Farzan博士和Hyeryun Choe博士的指导下进行，他们在病毒进入方面有着长期的专业知识，并且是识别病毒受体的先驱。本提案中的策略密切模仿了他们鉴定SARS冠状病毒和出血性新世界沙粒病毒受体以及HIV趋化因子共受体的方法。除了这些研究目标和实验之外，候选人的职业发展目标是获得基本病毒学，病毒发病机制，蛋白质结构-功能，高通量遗传筛选和研究伦理行为方面的专业知识。候选人将通过参加病毒学、蛋白质生物化学、遗传筛选策略和负责任的研究行为方面的一系列正式研讨会和纳米课程来实现这些直接目标。候选人还将通过NERCE BEID接受必要的BSL-3生物防护、生物安全和安保培训，以便在其核心设施中使用活的新世界甲病毒进行验证性实验。候选人的长期职业目标是获得终身教职，并继续他对新世界甲型病毒进入机制和发病机制的研究。候选人的近期研究培训目标和长期职业目标也将通过奖学金监督委员会的指导和指导得到促进，该委员会由一群杰出的科学家组成，他们具有与候选人研究领域相关的前沿专业知识。波士顿儿童医院和哈佛医学院的培训机会和资源，以及博士的指导。Farzan和Choe是他职业发展计划的理想环境。波士顿儿童医院致力于这一职业发展计划，并保证候选人将能够将至少85%的全职工作投入到本提案中所描述的活动中。

DESCRIPTION (provided by applicant): Eastern equine encephalitis virus (EEEV) is one of the most highly pathogenic viruses in humans with 35-50% mortality and is an actively reemerging public health threat. EEEV is a New World alphavirus, a group of mosquito-borne encephalitic pathogens with a wide geographical distribution and broad host range that includes the related agents Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV). A better understanding of the viral and host determinants of New World alphaviral entry into host cells will lend insight into critical aspects of the viral life cycle and pathogenicity and may provide a basis for the development of future treatment strategies and therapies. A molecular understanding of New World alphaviral tropism and entry has been limited by the requirement for special biosafety containment facilities. To overcome these limitations, we have developed "pseudotyped" viruses, which contain the EEEV, WEEV or VEEV envelope glycoproteins on their surface and express a reporter gene as a marker of infection. Each pseudotyped virus recapitulates the native entry mechanisms of the specific viruses on which they are based and can be used in standard BSL-2 conditions. We have also generated soluble New World alphaviral E2 glycoprotein ectodomains that bind permissive target cells. These reagents enable a comparative investigation into the molecular details that govern the viral entry pathways of representative and important New World alphaviruses. The goals of this proposal are to define the receptor binding domains within the New World alphaviral envelope glycoproteins, to determine if New World alphaviruses share a common cell surface receptor and to identify their specific host entry factors. In the first aim, the candidate will generate soluble forms of New World alphaviral E2 glycoproteins with iterative N and C-terminal truncations and site-directed mutations and characterize their ability to bind target cells. In the second aim, the candidate will comparatively characterize New World alphaviral tropism on a broad range of target cells, map common receptor usage through a series of binding and infection competitions, and identify specific cell surface receptors by immunoprecipitation with soluble alphaviral E2 receptor binding domains. Through the support of the Broad Institute, in the third aim the candidate will identify specific host cell entry factors by high-throughput genetic screening of human cells that have become non-permissive to New World alphaviral pseudotype infection using a shRNA library. The importance of potential host factors for New World alphaviruses identified with soluble receptor binding domains or using the genetic screen with pseudotyped viruses will be confirmed in experiments with live virus at the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE BEID) BSL-3 core facility. These studies will be conducted under the mentorship of Dr. Michael Farzan and Dr. Hyeryun Choe who have a long-standing expertise in viral entry and are pioneers in the identification of viral receptors. The strategies in this proposal are closely modeled after their approach towards the identification of the receptors for the SARS coronavirus and hemorrhagic New World arenaviruses and the chemokine co-receptors for HIV. In addition to these proposed research aims and experiments, the candidate's career development goals are to gain expertise in basic virology, viral pathogenesis, protein structure-function, high throughput genetic screens and the ethical conduct of research. The candidate will achieve these immediate goals by attending a series of formal seminars and nanocourses in virology, protein biochemistry, genetic screening strategies and the responsible conduct of research. The candidate will also receive the necessary BSL-3 biocontainment, biosafety, and security training through NERCE BEID to perform confirmatory experiments with live New World alphaviruses at their core facility. The candidate's long-term career goals are to attain a tenure-track faculty position and to continue his research on the mechanisms of New World alphaviral entry and pathogenesis. The candidate's immediate research training goals and long-term career goals will also be facilitated through the guidance and mentorship of his scholarship oversight committee composed of a distinguished group of scientists with leading-edge expertise pertinent to the candidate's field of study. The training opportunities and resources at Children's Hospital Boston and Harvard Medical School together with the mentorship of Drs. Farzan and Choe are an ideal environment for his career development program. Children's Hospital Boston is committed to this career development plan and has assured the candidate will be able to devote at least 85% full-time effort to the activities described in this proposal.