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

描述（由申请方提供）：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（虫媒病毒）复制的影响。自噬是介导细胞质物质转移到溶酶体进行降解的进化保守过程。自噬的诱导导致形成称为自噬体的双磷脂膜囊泡，其隔离靶向细胞器和蛋白质。随后，自噬体与介导其内容物降解的溶酶体融合。这个过程有助于维持细胞的稳态和生存。此外，自噬作为针对细胞内病原体（如结核分枝杆菌和刚地弓形虫）的先天免疫防御发挥作用。有趣的是，自噬被认为具有促病毒和抗病毒活性。因此，其在宿主-病毒相互作用中的作用目前尚不清楚。 登革病毒（DENV;黄病毒科，黄病毒）在世界范围内引起巨大的发病率和死亡率，并且控制的选择是有限的。因此，必须阐明影响病毒-载体相互作用的因素，以开发新的控制策略。先前的研究已经证明，自噬在哺乳动物细胞感染期间以pro-DENV能力起作用，但自噬在媒介蚊子的虫媒病毒感染期间的作用尚不清楚。因此，我们建议检查自噬在埃及伊蚊感染DENV-2期间的作用。我们假设自噬在DENV感染Ae期间以亲病毒能力运作。埃及人。 具体目标1在三种无脊椎动物细胞培养系统中解决了这个问题; Ae.白纹伊蚊C6/36、Ae.白纹伊蚊U4.4和果蝇S2细胞。作为目标1的补充，具体目标2将研究自噬在DENV感染Ae期间的作用。埃及人。此外，我们将阐明，如果自噬功能作为一个决定因素的Ae。使用远交蚊子品系的埃及伊蚊载体对DENV的感受态。自噬在病毒-载体相互作用中的作用将使用自噬的药理学抑制剂和诱导剂以及转基因蚊子进行分析。我们将使用三种常规测定法，电子显微镜，GFP-Atg-8斑点的共聚焦显微镜分析和通过免疫印迹定量脂化Atg-8- II来监测自噬的标志。此外，将使用标准病毒学测定来评估对DENV感染、复制和传播的影响。 我在病毒-载体相互作用方面有广泛的背景，并有必要的专业知识和动机来完成拟议的研究。我在媒介生物学和虫媒病毒学领域的研究有很好的记录，这从我的出版记录中可以看出。我以前的研究支持进一步证明了这一点：Kirschstein研究服务奖（5 F32 AI 084432 -02），题为“RNA干扰在西尼罗河病毒遗传多样性中的作用”。我一直在积极追求终身教职，最近在四个高度重视的机构面试。虽然我还没有收到录用通知，但这些邀请表明我是有竞争力的候选人。我的长期职业目标包括获得终身教职，在病媒生物学和虫媒病毒学领域建立独立和创新的研究项目，维持一个资金充足的实验室，并为科学界做出有意义的贡献，这些贡献有朝一日可能会在现实世界中得到应用。我对病媒生物学和虫媒病毒学的持续兴趣和热情，加上我的专业事业，证明了我在学术界获得教职的承诺。包括在这个应用程序是一个职业发展计划，将促进我的发展作为一个年轻的调查员，并帮助我实现我的长期目标。具体来说，我计划参加NIH的项目资助和赠款区域研讨会，这不仅会提高我的赠款写作技巧，而且还会让我了解联邦法规和政策，并让我了解当前特别感兴趣的领域。此外，我将参加美国病毒学学会和美国热带医学与卫生学会每年的会议，以及参加更亲密的基石研讨会。这些会议将使我能够更容易地与科罗拉多州立大学（CSU）以外的研究人员建立合作。职业发展超越了写作和研究。在年轻调查员的职业发展中，一个经常被忽视的方面是教学。CSU通过学习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，在我的博士后任期内获得了一些教学经验，我会利用这个研究所提供的资源，如教学与技术研讨会和教学设计咨询，以提高我的教学效果。拟议研究的新奇及其对病毒-载体相互作用的潜在影响，以及节肢动物生理学，以及我的职业发展计划和良好的业绩记录，使我成为K-22职业过渡奖的理想候选人。

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.