The role of autophagy in Aedes aegypti vector competence for dengue virus type 2

自噬在埃及伊蚊载体抵抗登革热病毒 2 型能力中的作用

• 批准号: 8510060
• 负责人: Douglas E. Brackney
• 金额: $ 16.2万
• 依托单位: CONNECTICUT AGRICULTURAL EXPERIMENT STA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-15 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510060
• 关键词: Academia; Address; Aedes; Affect; American; Animal Model; Antiviral Agents; Antiviral Response; Arboviruses; Area; Arthropods; Attention; Autophagocytosis; Autophagosome; Award; Biological Assay; Biology; Career Transition Award; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Survival; Cell physiology; Cells; Chemicals; Collaborations; Colorado; Communities; Competence; Complex; Confocal Microscopy; Consultations; Culicidae; Dengue Virus; Development; Development Plans; Direct Expenditure; Drosophila genus; Educational process of instructing; Educational workshop; Effectiveness; Electron Microscopy; Exhibits; Faculty; Flaviviridae; Flavivirus; Fostering; Funding; Genes; Goals; Grant; Hemocytes; Homeostasis; Human; Hygiene; Immune; Immunity; Immunoblotting; Infection; Institutes; Institution; Integration Host Factors; Interview; Invertebrates; Kinetics; Laboratories; Learning; Literature; Lysosomes; Mammalian Cell; Measures; Mediating; Membrane; Midgut; Monitor; Morbidity - disease rate; Mosquito Control; Motivation; Mycobacterium tuberculosis; Organelles; Pathway interactions; Phenotype; Phospholipids; Physiology; Policy Making; Population; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Public Health; Publications; RNA Interference; Regulation; Research; Research Personnel; Research Support; Resources; Rhabdoviridae; Role; Salivary Glands; Secure; Services; Societies; Staging; Technology; Therapeutic; Toxoplasma gondii; Transgenic Organisms; Tropical Medicine; Universities; Vaccines; Vesicle; Vesicular stomatitis Indiana virus; Vesiculovirus; Viral; Viral Load result; Virus; Virus Diseases; West Nile virus; Writing; career; career development; design; differential expression; disorder risk; experience; fly; human disease; improved; inhibition of autophagy; inhibitor/antagonist; innovation; interest; meetings; mortality; novel; novel strategies; pathogen; programs; protein aggregate; real world application; skills; symposium; transmission process; vector; vector mosquito; virology; virus genetics; virus host interaction

DESCRIPTION (provided by applicant): The overarching objective of this application is to investigate the influence of autophagy on the replication of arthropod-borne viruses (arboviruses) in invertebrates. Autophagy is an evolutionarily conserved process that mediates the transfer of cytoplasmic material to lysosomes for degradation. Induction of autophagy results in the formation of double-phospholipid membrane vesicles termed autophagosomes which sequester targeted organelles and proteins. Subsequently, autophagosomes fuse with lysosomes which mediate degradation of their contents. This process helps maintain cellular homeostasis and survival. In addition, autophagy functions as an innate immune defense against intracellular pathogens, such as Mycobacterium tuberculosis and Toxoplasma gondii. Interestingly, autophagy has been implicated as having both pro- and antiviral activity. Its role i host-virus interactions is therefore unclear at present. Dengue virus (DENV; Flaviviridae, Flavivirus) causes tremendous morbidity and mortality worldwide, and options for control are limited. Therefore, it is imperative to elucidate factors influencing virus-vector interactions in order to develop novel control strategies. Previous studies have demonstrated that autophagy functions in a pro-DENV capacity during infection of mammalian cells, but the role of autophagy during arbovirus infection of vector mosquitoes is unknown. Therefore, we propose to examine the role of autophagy during DENV-2 infection of Aedes aegypti. We hypothesize that autophagy operates in a pro-viral capacity during DENV infection of Ae. aegypti. Specific aim 1 addresses this question in three invertebrate cell culture systems; Ae. albopictus C6/36, Ae. albopictus U4.4, and Drosophila S2 cells. Complementary to aim 1, specific aim 2 will investigate the role of autophagy during DENV infection of Ae. aegypti. In addition, we will elucidate if autophagy functions as a determinant of Ae. aegypti vector competence for DENV using outbred mosquito strains. The role of autophagy in the virus-vector interaction will be analyzed using pharmacological inhibitors and inducers of autophagy as well as transgenic mosquitoes. We will monitored for hallmarks of autophagy using three conventional assays, electron microscopy, confocal microscopy analysis of GFP-Atg-8 punctae, and quantification of lipidated Atg-8- II by immunoblot. Further, the effects on DENV infection, replication and transmission will be assessed using standard virological assays. I have an extensive background in virus-vector interactions and have the necessary expertise and motivation to complete the proposed studies. I have a strong track record of research in the fields of vector biology and arbovirology as evident from my publication record. This is further exemplified by my former research support: a Ruth L. Kirschstein Research Service Award (5F32AI084432-02) entitled "The Role of RNA Interference in West Nile virus Genetic Diversification". I have been actively pursuing tenure-track faculty positions and have recently interviewed at four highly regarded institutions. While I have yet to receive an offer, these invitations indicate that I am competitive candidate. My long-term career goals include securing a tenure-track faculty position, establishing an independent and innovative research program in vector biology and arbovirology, maintaining a well-funded laboratory, and making meaningful contributions to the scientific community that may, someday, have real-world applications. My continued interest and passion for vector biology and arbovirology combined with my professional undertakings attest to my commitment to secure a faculty position in academia. Included in this application is a career development plan that will foster my development as a young investigator and help me achieve my long-term goals. Specifically, I plan on attending the NIH's Regional Seminars on Program Funding and Grants which will not only improve my grant writing skills, but also inform me of federal regulations and policies and make me aware of current areas of special interest. Furthermore, I will be attending the American Society of Virology and the American Society of Tropical Medicine and Hygiene conferences annually as well as attending more intimate Keystone Symposia. These meetings will allow me to more easily establish collaborations with researchers outside of Colorado State University (CSU). Career development extends beyond grant writing and research. An often overlooked aspect in the career development of young investigators is teaching. CSU offers numerous courses through the Institute for Learning and Teaching dedicated to improving the teaching skills of faculty and staff. While I have been fortunate to acquire some teaching experience during my post-doc tenure, I will take advantage of the resources available through this institute such as Teaching with Technology workshops and Instructional Design Consultation to improve my teaching effectiveness. The novelty of the proposed research and its potential impact on not only virus-vector interactions, but also arthropod physiology as well as my career development plan and strong track record make me an ideal candidate for the K-22 Career Transition Award.

描述（由申请人提供）：本申请的总体目标是研究自噬对无脊椎动物中节肢动物传播病毒（Arbovirus）复制的影响。自噬是一种进化保守的过程，可介导细胞质材料向溶酶体的转移以降解。自噬的诱导会导致形成称为自噬体的双磷脂膜囊泡，这些囊泡隔离了靶向细胞器和蛋白质。随后，自噬体与介导其含量降解的溶酶体融合。这个过程有助于维持细胞稳态和生存。此外，自噬是针对细胞内病原体的先天免疫防御，例如结核分枝杆菌和弓形虫弓形虫。有趣的是，自噬已被暗示为具有促病毒活性。因此，目前尚不清楚其作用I主机病毒相互作用。 登革热病毒（DENV； Flaviviridae，Flavivivirus）在全球范围内引起巨大的发病率和死亡率，并且控制选择受到限制。因此，必须阐明影响病毒载体相互作用的因素，以制定新颖的控制策略。先前的研究表明，自噬功能在哺乳动物细胞感染过程中具有促粉末能力的功能，但是自噬在弧病毒感染中自噬的作用尚不清楚。因此，我们建议检查自噬在伊德斯伊德斯伊德斯（Aedes Aegypti）的DENV-2感染中的作用。我们假设自噬在AE的DENV感染过程中以促病毒的能力运行。埃及。 特定目的1在三个无脊椎动物细胞培养系统中解决了这个问题。 Ae。白化病C6/36，AE。 Alboptus U4.4和果蝇S2细胞。 AIM 1的补充，特定的目标2将研究自噬在AE的DENV感染中的作用。埃及。另外，我们将阐明自噬是否作为AE的决定因素。使用杂种蚊子菌株的DENV的埃及载体载体能力。自噬在病毒载体相互作用中的作用将使用自噬和转基因蚊子的药理抑制剂和诱导剂进行分析。我们将使用三种常规测定法，电子显微镜，GFP-ATG-8点的共聚焦显微镜分析以及通过免疫印迹对脂质ATG-8-II进行定量来监测自噬的标志。此外，将使用标准病毒学测定法评估对DENV感染，复制和传播的影响。 我在病毒载体相互作用方面具有广泛的背景，并具有完成拟议研究的必要专业知识和动力。从我的出版记录中可以明显看出，我在媒介生物学和杂植物学领域的研究有很强的记录。我以前的研究支持进一步说明了这一点：露丝·柯希斯坦（Ruth L. Kirschstein）研究服务奖（5F32AI084432-02），名为“ RNA干扰在西尼罗河遗传多样化中的作用”。我一直在积极担任终身教师职位，最近在四个备受推崇的机构中接受了采访。尽管我尚未收到要约，但这些邀请表明我是竞争性候选人。我的长期职业目标包括确保统治轨道的教职员工，建立独立和创新的研究计划，领域的生物学和arbovirology，维持资金充足的实验室，并为科学社区做出有意义的贡献 - 世界应用程序。我对媒介生物学和杂技病理学的持续兴趣和热情加上我的专业事业证明了我致力于确保学术界职位的承诺。本申请中包括一项职业发展计划，该计划将促进我作为年轻调查员的发展，并帮助我实现长期目标。具体来说，我计划参加NIH的有关计划资金和赠款的区域研讨会，这不仅可以提高我的赠款写作技巧，而且还将告知我联邦法规和政策，并使我意识到当前的特殊兴趣领域。此外，我将每年参加美国病毒学和美国热带医学与卫生会议学会，并参加更亲密的Keystone专题讨论会。这些会议将使我更容易与科罗拉多州立大学（CSU）以外的研究人员建立合作。职业发展不仅仅是赠款写作​​和研究。年轻调查人员的职业发展中经常被忽视的方面是教学。 CSU通过研究和教学研究所提供许多课程，致力于提高教职员工的教学技能。虽然我很幸运能够在工作后任职期间获得一些教学经验，但我将利​​用该学院可用的资源，例如在技术研讨会和教学设计咨询中进行教学，以提高我的教学效果。拟议研究的新颖性及其对病毒武器相互作用的潜在影响，而且对节肢动物的生理学以及我的职业发展计划和强大的往绩使我成为K-22职业过渡奖的理想候选人。