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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我于2008年7月加入了UVM的教师队伍，目前正在完成我第二年的COBRE-supported研究。在过去的一年里，我把我的努力集中在推进我的研究计划，准备出版的手稿，并编写赠款建议，以获得未来研究的校外资金。 我的研究项目主要集中在新兴传染病，特别是汉坦病毒和沙粒病毒。在前一年的资助中，我们利用了最先进的蛋白质组学方法来鉴定人ER-高尔基体中间室-53 kDa蛋白（ERGIC-53）作为安第斯山脉汉坦病毒（ANDV）编码的糖蛋白前体（GPC）的潜在相互作用伴侣。因此，我们在本供资期间非常详细地研究了这种相互作用。为了独立地验证相互作用，我们从人细胞中亲和纯化ANDV GPC，并发现ERGIC-53通过Western印迹共免疫沉淀。我们还进行了相互实验，发现ERGIC-53的亲和纯化共免疫纯化ANDV GPC。为了确定这种相互作用是否是高度保守的，我们测试了由其他汉坦病毒以及沙粒病毒编码的GPC是否也与ERGIC-53相互作用。有趣的是，我们发现来自Sin Nombre汉坦病毒以及几种致病性沙粒病毒（拉沙病毒（LASV），朱宁病毒（JUNV），淋巴细胞性脉络丛脑膜炎病毒（LCMV），Machupo病毒和白水阿罗约病毒）的GPC也与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沉默人细胞中的ERGIC-53表达，然后用JUNV和LCMV攻击这些细胞，以确定ERGIC-53的缺失如何影响每种病毒进行生产性复制的能力。与用乱序siRNA转染的对照细胞相比，我们观察到JUNV和LCMV的ERGIC-53敲低后病毒滴度显著降低。因此，我们发现在JUNV攻击之前细胞中ERGIC-53的过表达导致病毒滴度的显著增加。ERGIC-53最初被发现作为凝血因子V和VIII的货物受体的重要作用; ERGIC-53突变的个体由于不能分泌因子V和VIII而患有出血性疾病。ERGIC-53可能是沙粒病毒和汉坦病毒GPCs从ER有效转运至高尔基体所需的货物受体。我们的研究结果表明，ERGIC-53在沙粒病毒复制中起着重要作用，因此可能是开发广谱抗病毒药物的有价值的靶标，以靶向致病性沙粒病毒，也可能靶向汉坦病毒。另一个有趣的假设是ERGIC-53与JUNV、LASV或ANDV编码的GPC的相互作用可能破坏其对凝血因子V和VIII的正常伴侣功能，导致感染这些病毒后观察到的出血表现。我们目前正在准备一份手稿，描述我们的结果提交给PLoS病原体。明年，我们计划研究ERGIC-53与病毒GPCs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形态发生和ERGIC中病毒工厂的形成，并确定ERGIC-53对细胞蛋白（包括因子V和VIII）的正常货物功能是否通过与沙粒病毒和汉坦病毒GPC的相互作用而受损。这些拟议的研究将成为RO 1申请的主题，该申请将提交给NIAID。