HANTAVIRUS AND ARENAVIRUS HOST-PATHOGEN INTERACTIONS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我于2008年7月加入密歇根州立大学的教职员工队伍，目前正在完成科布雷资助的研究的第二年。在过去的一年里，我集中精力推进我的研究计划，准备出版手稿，并撰写拨款提案，为未来的研究获得外部资金。 我的研究项目专注于新出现的传染病，特别是汉坦病毒和阿拉伯病毒。在前一年的资助中，我们利用最先进的蛋白质组学方法确定了人类ER-高尔基中间区-53 kDa蛋白(ERGIC-53)是安第斯汉坦病毒(ANDV)编码的糖蛋白前体(GPC)的潜在相互作用伙伴。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了ANDV GPC，并通过Western印迹发现ERGIC-53是免疫共沉淀的。我们还进行了相互实验，发现ERGIC-53的亲和纯化共免疫吸附ANDV GPC。为了确定这种相互作用是否高度保守，我们测试了由其他汉坦病毒和Arena病毒编码的GPC是否也与ERGIC-53相互作用。有趣的是，我们发现Sin Norbre汉坦病毒的GPC以及几种致病Arena病毒(Lassa病毒(LASV)、Junin病毒(JUNV)、淋巴细胞性脉络膜脑膜炎病毒(LCMV)、Machupo病毒和白水Arroyo病毒)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沉默了ERGIC-53在人类细胞中的表达，然后用JUNV和LCMV挑战这些细胞，以确定ERGIC-53的缺失将如何影响每种病毒进行生产性复制的能力。与对照组细胞相比，我们观察到ERGIC-53下调JUNV和LCMV的病毒滴度后，病毒滴度显著降低。相反，我们发现在JUNV攻击前ERGIC-53在细胞中的过表达导致病毒滴度显著增加。ERGIC-53最初被发现是因为它作为凝血因子V和VIII的货物受体的重要作用；ERGIC-53突变的个体由于无法分泌V和VIII因子而患有出血性疾病。可能是ERGIC-53是一种货物受体，需要有效地将Arena病毒和汉坦病毒GPC从ER运送到高尔基体。我们的研究结果表明，ERGIC-53在阿拉伯病毒复制中发挥着重要作用，因此可能是开发广谱抗病毒药物的有价值的靶点，以针对致病的阿拉伯病毒，并潜在地针对汉坦病毒。另一个有趣的假说是，ERGIC-53‘S与JUNV、LASV或ANDV编码的GPC的相互作用可能会扰乱其对凝血因子V和VIII的正常伴侣功能，导致感染这些病毒后出现的出血表现。我们目前正在准备一份手稿，描述我们的结果，以便提交给PLoS病原体。明年，我们计划研究ERGIC-53与病毒GPC相互作用的几个方面。具体地说，我们计划定义这种相互作用的分子基础，确定这种相互作用如何促进GPC的形态发生和ERGIC中病毒工厂的形成，并确定ERGIC-53‘S对细胞蛋白(包括因子V和VIII)的正常运输功能是否因与禽源病毒和汉坦病毒GPC的相互作用而受损。这些拟议的研究将成为RO1申请的主题，该申请将被汇编并提交给NIAID。

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.