Viral and Host Determinants of New World Alphaviral Entry

新世界甲病毒进入的病毒和宿主决定因素

• 批准号: 8090665
• 负责人: Asim Aminsharif Ahmed
• 金额: $ 13.35万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090665
• 关键词: Affinity; Alphavirus; Arenavirus; Binding; Biochemical; Biochemical Genetics; Biological Assay; Boston; Brain; C-terminal; Candidate Disease Gene; Cell Surface Receptors; Cell-Matrix Junction; Cells; Clathrin; Commit; Complement; Containment; Core Facility; Culicidae; Development; Development Plans; Eastern Equine Encephalitis Virus; Emerging Communicable Diseases; Environment; Ethics; Faculty; Future; Genes; Genetic; Genetic Screening; Genetic screening method; Glycoproteins; Goals; HIV Receptors; Human; Immunoprecipitation; Infection; Institutes; Integral Membrane Protein; Integration Host Factors; Integrins; Investigation; Laboratories; Lectin; Libraries; Life; Life Cycle Stages; Link; Maps; Mentors; Mentorship; Methods; Modeling; Modification; Molecular; Morbidity - disease rate; Mutation; New England; Pathogenesis; Pathogenicity; Pathway interactions; Pediatric Hospitals; Point Mutation; Positioning Attribute; Protein Biochemistry; Public Health; Reagent; Receptor Cell; Recombinants; Relative (related person); Reporter Genes; Research; Research Training; Resources; Role; SARS coronavirus; Scholarship; Scientist; Security; Series; Site; Surface; Tacaribe Complex Viruses; Therapeutic; Time; Training; Tropism; Venezuelan Equine Encephalitis Virus; Viral; Viral Hemorrhagic Fevers; Viral Pathogenesis; Virulence; Virus; Virus Diseases; Virus Receptors; Western Equine Encephalitis Virus; Work; base; biodefense; career; career development; cell type; chemokine; comparative; field study; glycosylation; insight; medical schools; mortality; particle; pathogen; programs; protein structure function; public health relevance; receptor; receptor binding; research study; responsible research conduct; small hairpin RNA; treatment strategy; virology; virus envelope

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.

描述(申请人提供)：东部马脑炎病毒(EEEV)是人类最高致病性病毒之一，死亡率为35%-50%，是一种正在重新出现的公共卫生威胁。EEEV是一种新型世界甲型病毒，是一组地理分布广、宿主范围广的蚊媒脑炎病原体，包括西方马脑炎病毒(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，并可能为未来治疗战略和治疗的发展提供基础。由于需要特殊的生物安全控制设施，对新世界甲型病毒嗜性和进入的分子理解一直受到限制。为了克服这些局限性，我们开发了“假型”病毒，它们的表面含有EEEV、WeEV或VEEV包膜糖蛋白，并表达一个报告基因作为感染的标志。每一种伪型病毒都概括了它们所基于的特定病毒的本地入侵机制，并可在标准的BSL-2条件下使用。我们还产生了可溶的新世界甲型病毒E2糖蛋白胞外区，与允许的靶细胞结合。这些试剂能够对控制典型和重要的新大陆甲型病毒进入病毒途径的分子细节进行比较研究。该方案的目的是确定新世界甲型病毒囊膜糖蛋白中的受体结合域，确定新世界甲型病毒是否共享共同的细胞表面受体，并确定其特定的宿主进入因子。在第一个目标中，候选者将产生具有迭代N端和C端截断和定点突变的可溶形式的新世界甲型病毒E2糖蛋白，并鉴定它们结合靶细胞的能力。在第二个目标中，候选人将比较表征新世界甲型病毒在广泛的靶细胞上的嗜性，通过一系列结合和感染竞争绘制共同受体的使用图，并通过免疫沉淀与可溶性甲型病毒E2受体结合域鉴定特定的细胞表面受体。通过博德研究所的支持，在第三个目标中，候选人将通过使用shRNA文库对已成为不允许新世界甲型病毒假型感染的人类细胞进行高通量遗传筛选，从而确定特定的宿主细胞进入因子。在新英格兰地区生物防御和新发传染病卓越中心(NERCE BEID)BSL-3核心设施进行的活病毒实验中，将确认利用可溶受体结合域或使用带有伪型病毒的遗传筛选确定的新世界甲型病毒潜在宿主因素的重要性。这些研究将在迈克尔·法赞博士和海伦·崔博士的指导下进行，他们在病毒进入方面拥有长期的专业知识，是识别病毒受体的先驱。这项建议中的战略是紧密仿照它们识别SARS冠状病毒和出血性新世界新冠病毒受体以及艾滋病毒趋化因子辅助受体的方法。除了这些拟议的研究目标和实验，候选人的职业发展目标是获得基础病毒学、病毒发病机制、蛋白质结构功能、高通量基因筛选和研究伦理方面的专业知识。候选人将通过参加一系列关于病毒学、蛋白质生物化学、基因筛查策略和负责任的研究工作的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生物遏制、生物安全和安全培训，以在其核心设施进行活的新世界甲型病毒的验证性实验。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他的奖学金监督委员会的指导和指导而得到促进，该委员会由一群具有与候选人研究领域相关的前沿专业知识的杰出科学家组成。波士顿儿童医院和哈佛医学院的培训机会和资源，再加上Farzan和Choe博士的指导，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，并向应聘者保证能够将至少85%的全职精力投入到这份建议书中描述的活动中。