HANTAVIRUS AND ARENAVIRUS HOST-PATHOGEN INTERACTIONS

汉坦病毒和沙粒病毒宿主-病原体相互作用

• 批准号: 8360777
• 负责人: Jason W. Botten
• 金额: $ 24.85万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-20 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360777
• 关键词: Affinity; Affinity Chromatography; Antiviral Agents; Applications Grants; Arenavirus; Bolivian Hemorrhagic Fever Virus; Cell physiology; Cells; Centers of Research Excellence; Communicable Diseases; Development; Disease; Emerging Communicable Diseases; Extramural Activities; Factor V; Faculty; Funding; Future; Glycoproteins; Golgi Apparatus; Grant; Hantavirus; Hemorrhage; Human; Individual; Infection; Junin virus; Lassa virus; Lymphocytic choriomeningitis virus; Manuscripts; Molecular; Molecular Chaperones; Morphogenesis; Mutation; National Center for Research Resources; National Institute of Allergy and Infectious Disease; Play; Principal Investigator; Proteins; Proteomics; Publications; Research; Research Infrastructure; Research Support; Resources; Role; Small Interfering RNA; Source; Testing; United States National Institutes of Health; Vermont; Viral; Viral Proteins; Virus; Western Blotting; Writing; base; cost; interest; knock-down; overexpression; pathogen; programs; protein expression; receptor; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. I joined the faculty at UVM in July of 2008, and am currently completing my second year of COBRE-supported research. In the past year, I have focused my efforts on advancing my research program, preparing manuscripts for publication, and writing grant proposals to obtain extramural funding for future studies. My research program is focused on emerging infectious diseases, particularly the hantaviruses and arenaviruses. In the previous year of funding, we had utilized a cutting edge proteomics approach to identify human ER-Golgi intermediate compartment-53 kDa protein (ERGIC-53) as a potential interacting partner of the glycoprotein precursor (GPC) encoded by Andes hantavirus (ANDV). Accordingly, we studied this interaction in great detail during the current funding period. To independently validate the interaction, we affinity purified, from human cells, the ANDV GPC and found that ERGIC-53 was co-immunoprecipated via Western blot. We also performed the reciprocal experiment and found that affinity purification of ERGIC-53 co-immunoprecipiated the ANDV GPC. To determine whether this interaction is highly conserved, we tested whether GPCs encoded by additional hantaviruses, as well as arenaviruses, also interact with ERGIC-53. Interestingly, we found that GPCs from Sin Nombre hantavirus, as well as several pathogenic arenaviruses (Lassa virus (LASV), Junin virus (JUNV), lymphocytic choriomeningitis virus (LCMV), Machupo virus, and Whitewater Arroyo virus), also interact with ERGIC-53. To determine the importance of ERGIC-53 for viral replication, we silenced ERGIC-53 expression in human cells via siRNA and then challenged these cells with JUNV and LCMV to determine how the absence of ERGIC-53 would impact the ability of each virus to undergo productive replication. Compared to control cells that were transfected with a scrambled siRNA, we observed a significant reduction in viral titer following ERGIC-53 knock-down for both JUNV and LCMV. Inversely, we found that overexpression of ERGIC-53 in cells prior to JUNV challenge led to a significant increase in viral titer. ERGIC-53 was originally discovered for its important role as a cargo receptor for the blood coagulation factors V and VIII; individuals with mutations in ERGIC-53 have bleeding disorders due to an inability to secrete factors V and VIII. It may be that ERGIC-53 is a cargo receptor required for the efficient transport of the arenavirus and hantavirus GPCs from the ER to the Golgi. The results of our studies suggest that ERGIC-53 plays an important role in arenavirus replication and may therefore represent a valuable target for the development of broad-spectrum antivirals to target the pathogenic arenaviruses and, potentially, the hantaviruses as well. Another interesting hypothesis is that ERGIC-53's interaction with the GPCs encoded by JUNV, LASV, or ANDV may disrupt its normal chaperone function for the blood coagulation factors V and VIII, leading to the hemorrhagic manifestations seen following infection with these viruses. We are currently preparing a manuscript describing our results for submission to PLoS Pathogens. In the next year, we plan to study several aspects of the ERGIC-53 interaction with viral GPCs. Specifically, we plan to define the molecular basis for the interaction, determine how the interaction contributes to GPC morphogenesis and the formation of viral factories in the ERGIC, and to determine whether ERGIC-53's normal cargo function for cellular proteins, including the factors V and VIII, is impaired via interaction with arenavirus and hantavirus GPCs. These proposed studies will be the subject of an RO1 application that will be assembled for submission to NIAID.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我于2008年7月加入UVM的教职员工，目前正在完成我第二年的鞋垫研究。在过去的一年中，我一直致力于推进研究计划，准备出版的手稿以及编写赠款建议，以获得未来研究的校外资金。 我的研究计划的重点是新兴的传染病，尤其是汉坦病毒和体育症病毒。在上一年的资金中，我们利用了一种最先进的蛋白质组学方法来鉴定人类ER-Golgi中间室-53 kDa蛋白（ERGIC-53）作为由Andes Hantavirus（Andes Hantavirus（Andv）编码的糖蛋白前体（GPC）的潜在相互作用伴侣。因此，我们在当前的资金期间对这种互动进行了详细的研究。为了独立验证这种相互作用，我们从人类细胞中纯化了ANDV GPC，并发现Ergic-53是通过Western blot共免疫沉淀的。我们还进行了倒数实验，发现Ergic-53 co-munifectection的亲和力纯化对ANDV GPC进行了纯化。为了确定这种相互作用是否高度保守，我们测试了GPC是否由其他汉坦病毒编码以及体轨道病毒也与ERGIC-53相互作用。有趣的是，我们发现来自Sin nombre Hantavirus的GPC以及几种致病性体育症（Lassa Virus（LASV），Junin病毒（JUNV），淋巴细胞性脉络膜脑膜炎病毒（LCMV），Machupo Virus病毒，以及与Ergic-53相互作用。为了确定ERGIC-53对病毒复制的重要性，我们通过siRNA在人类细胞中沉默了Ergic-53表达，然后用JUNV和LCMV挑战了这些细胞，以确定不存在ERGIC-53如何影响每个病毒进行生产性复制的能力。与用炒siRNA转染的对照细胞相比，我们观察到JunV和LCMV均可敲击ERGIC-53敲低后病毒滴度的显着降低。相反，我们发现在JUNV挑战之前的细胞中Ergic-53的过表达导致病毒滴度显着增加。 Ergic-53最初是因为其作为血液凝结因子V和VIII的货物受体的重要作用而被发现的。 ERGIC-53中突变的个体由于无法分泌VICH和VIII而患有出血性疾病。可能是Ergic-53是一种有效运输体体和Hantavirus GPC从ER到Golgi所需的货​​物受体。我们的研究结果表明，ERGIC-53在体育症病毒复制中起着重要作用，因此可能代表着开发广谱抗病毒药的有价值的靶标，以靶向致病病毒，并可能是汉坦病毒。另一个有趣的假设是，Ergic-53与JUNV，LASV或ANDV编码的GPC的相互作用可能会破坏其在血液凝结因子V和VIII中的正常伴侣功能，从而导致通过这些病毒感染后观察到的出血性表现。我们目前正在准备一个手稿，描述我们提交PLOS病原体的结果。明年，我们计划研究Ergic-53与病毒GPC的相互作用的几个方面。具体而言，我们计划定义相互作用的分子基础，确定相互作用如何有助于GPC的形态发生和ERGIC中病毒工厂的形成，并确定Ergic-53的正常货物对细胞蛋白的正常功能，包括因素V和VIII，包括因子V和VIII，通过与Arenavirus andavirus gpcs互动而受到损害。这些拟议的研究将是RO1应用程序的主题，该申请将组装出来，以提交给NIAID。