Identification of Novel Arenavirus Protein-Host Cellular Protein Interactions

描述(申请人提供)：ArenaVirus是一种负义RNA病毒家族，可导致从无菌性脑膜炎到出血热综合征等严重的人类疾病。ArenaVirus蛋白质组只由四种蛋白质组成：小环指蛋白Z、核蛋白、病毒RNA聚合酶和糖蛋白前体，糖蛋白前体经过翻译后修饰产生异源二聚体包膜糖蛋白GP1和GP2。虽然这些蛋白在病毒复制中的功能作用已经明确，但它们与宿主细胞蛋白的相互作用以及这些相互作用对病毒复制和疾病发病机制的重要性在很大程度上是未知的。识别对病毒复制和/或病毒致病至关重要的新的病毒蛋白-宿主蛋白相互作用将促进我们对这些NIAID A类病毒的基本生物学的了解，并为抗病毒药物的开发提供新的靶点。因此，我们将使用先进的蛋白质组学方法，其特点是亲和纯化与宿主细胞蛋白形成的复合体中的病毒蛋白，以及多维质量谱蛋白质鉴定技术(MudPIT)，以确定与选定的旧世界(淋巴细胞性脉络膜脑膜炎病毒)和新世界(Junin病毒)致病Arena病毒的蛋白质组相互作用的宿主蛋白质伙伴。我们的团队最近利用这种方法成功地鉴定了几种与病毒蛋白相互作用的宿主蛋白，包括在汉坦病毒和阿雷纳病毒之间保守的相互作用。此外，我们已经产生了这项工作所需的必要试剂，即在哺乳动物细胞中表达由六种对人类致病的Arena病毒编码的每个ORF的质粒载体文库。通过这项工作确定的ArenaVirus蛋白-宿主蛋白相互作用将为未来的赠款申请提供基础，以更全面地描述这些相互作用，特别是它们对病毒复制和致病机制的影响。作为确定未来研究中最相关的相互作用的初步手段，我们将通过在选择性击倒宿主蛋白伙伴后测量允许细胞系中的病毒复制来评估已识别的ArenaVirus蛋白-宿主蛋白相互作用对病毒复制的重要性。 公共卫生相关性：阿雷纳病毒导致严重的人类疾病，从无菌性脑膜炎到出血热综合征。这些病毒产生四种蛋白质。该项目的目标是确定这些ArenaVirus蛋白在感染过程中与之相互作用的人类宿主蛋白，以便我们更好地了解这些病毒蛋白-宿主蛋白相互作用如何影响病毒的复制能力和导致人类疾病。重要相互作用的识别将为抗病毒药物的开发提供新的靶点。

DESCRIPTION (provided by applicant): The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. The arenavirus proteome consists of only four proteins: the small RING finger protein Z, nucleoprotein, viral RNA polymerase, and glycoprotein precursor, which is post-translationally modified to yield the heterodimeric envelope glycoproteins GP1 and GP2. While the functional role of these proteins for viral replication is well defined, their interactions with host cellular proteins, and the importance of these interactions for viral replication and disease pathogenesis, is largely unknown. Identification of novel arenavirus protein-host protein interactions that are critical for viral replication and/or viral pathogenesis would advance our understanding of the basic biology of these NIAID Category A viruses and provide new targets for the development of antivirals. Accordingly, we will use a cutting edge proteomics approach that features affinity purification of viral proteins in complex with host cellular proteins and multi-dimensional mass spectrometry protein identification technology (MudPIT) to identify host protein partners that interact with the proteomes of selected Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) pathogenic arenaviruses. Our group has recently utilized this approach to successfully identify several host proteins that interact with viral proteins, including interactions that are conserved among both hantaviruses and arenaviruses. In addition, we have already generated the necessary reagents required for this work, namely a library of plasmid vectors that express, in mammalian cells, each of the ORFs encoded by six arenaviruses that are pathogenic for humans. The arenavirus protein-host protein interactions identified through this work will provide the basis for future grant applications to characterize these interactions more fully, particularly their impact on viral replication and pathogenesis. As an initial means to determine which interactions would be most relevant to pursue in future studies, we will evaluate the importance of the identified arenavirus protein-host protein interactions for viral replication by measuring viral replication in permissive cell lines following selective knock-down of host protein partners. PUBLIC HEALTH RELEVANCE: The arenaviruses cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. These viruses produce four proteins. The goal of this project is to identify the human host proteins that these arenavirus proteins interact with during infection so that we can better understand how these virus protein-host protein interactions impact both the ability of the virus to replicate and cause human disease. Identification of important interactions will provide new targets for the development of antivirals.