Characterization of the La Crosse Virus glycoprotein fusion peptide

拉克罗斯病毒糖蛋白融合肽的表征

基本信息

批准号：
7624319
负责人：
Francisco Gonzalez-Scarano
金额：
$ 34.78万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7624319
关键词：
Aedes Africa Alphavirus Amino Acids Antibodies Arbovirus Encephalitis Area Aseptic Meningitis Attenuated Bunyamwera virus Bunyaviridae Categories Cell fusion Cells Childhood Chimeric Proteins Clinical Collaborations Computer Analysis Crimean-Congo Hemorrhagic Fever Virus Culicidae Cysteine Encephalitis Epidemic Family Flavivirus Genome Genus Alpharetrovirus Glycoproteins Herpes encephalitis Immunity In Vitro Incidence Infection Insecta Knowledge La Crosse virus Laboratories Location Mammalian Cell Maps Mediating Membrane Midwestern United States Modeling Mus Mutagenesis Mutation National Institute of Allergy and Infectious Disease Neuraxis Neuropathogenesis Ochlerotatus Orthobunyavirus Peptide antibodies Peptides Phenotype Play Process Proteins RNA Recombinants Recurrence Reporting Rift Valley fever virus Role Sin Nombre virus Sindbis Virus Structural Protein System Technology Tissues Vaccines Viral Virus Virus Diseases Work base design epizootic flavivirus glycoprotein E flaviviruses glycoprotein E inhibitor/antagonist member mouse model mutant neurovirulence neutralizing antibody pathogen positional cloning public health relevance therapeutic development vector mosquito virus pathogenesis

项目摘要

DESCRIPTION (provided by applicant): La Crosse virus (LACV), a NIAID Category B priority pathogen, is a common cause of pediatric encephalitis and aseptic meningitis in areas of the Midwestern United States where its principal mosquito vector, Ochlerotatus (formerly Aedes) trisariatus, resides. The structural components of the LACV genome (L, M, and S) have essential, well- defined roles in virus pathogenesis. Previous studies from our laboratory using wild-type LACV and TAHV 181/57, a highly neurovirulent strain with low neuroinvasiveness, have mapped the neuroinvasive phenotype to the M segment, which encodes Gn, Gc, and a non-structural protein, NSm. More recently, using recombinant glycoproteins we demonstrated that the region corresponding to the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating fusion and cell entry. Further computational analysis identified structural similarities between LACV Gc amino acid region 970-1350 and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Collectively, these studies suggested that the LACV Gc, like the alphavirus E1 and the flavivirus E, functions as a type II fusion protein. Within Gc there is a 22 amino acid hydrophobic segment, 1066-1087, that is predicted to correlate structurally with a hydrophobic domain of SFV and Sindbis virus E1. The short sequence is highly conserved within the family Bunyaviridae and features several conserved cysteine residues, as do other type II proteins, such as SFV E1. Based on these features, and in our mutagenesis studies, our working hypothesis is that the LACV Gc (1066-1087) functions as its fusion peptide. In the first specific aim, we will extend these studies by analyzing fusion in mosquito cells, and by identifying peptides and antibodies that further associate this region with fusion and entry. In the second specific aim, we will use a newly developed reverse genetics system to construct LACV mutants incorporating the knowledge gained from the studies on the isolated glycoproteins. These viruses will then allow us to extend our in vitro findings to a mouse model of LACV encephalitis previously developed by our group (third specific aim). Importantly, as this hydrophobic region is highly conserved among the Bunyaviridae, this proposal will also elucidate mechanisms of virus fusion and entry among other emerging bunyaviruses including the NIAID Category A and C pathogens CCHFV and RVFV and will have significant implications for anti-viral therapy. PUBLIC HEALTH RELEVANCE La Crosse Virus is a common cause of pediatric encephalitis and aseptic meningitis in the Midwestern United States where it principal mosquito vector, Ochlerotatus triseriatus resides. We have identified the fusion peptide for the La Crosse virus glycoprotein Gc. The studies outlined in this proposal will define mechanisms of fusion and entry for La Crosse Virus in both the mammalian and insect host, determine the role of the newly identified fusion domain in the neuropathogenesis of LACV encephalitis, and develop anti-viral therapies (fusion peptide inhibitors and attenuated virus vaccines).

描述（由申请人提供）：拉克罗斯病毒 (LACV) 是 NIAID B 类优先病原体，是美国中西部地区小儿脑炎和无菌性脑膜炎的常见原因，该地区的主要蚊媒为三色赭蚊 (Ochlerotatus)（以前称为伊蚊），驻留。 LACV 基因组的结构成分（L、M 和 S）在病毒发病机制中具有重要且明确的作用。我们实验室之前使用野生型 LACV 和 TAHV 181/57（一种具有低神经侵袭性的高神经毒力菌株）进行的研究已将神经侵袭表型映射到 M 片段，该片段编码 Gn、Gc 和非结构蛋白 NSm。最近，使用重组糖蛋白，我们证明了对应于 Gc 近膜三分之二的区域（氨基酸 860-1442）对于介导融合和细胞进入至关重要。进一步的计算分析确定了 LACV Gc 氨基酸区域 970-1350 和两种甲病毒的 E1 融合蛋白之间的结构相似性：辛德比斯病毒和塞姆利基福雷斯特病毒 (SFV)。总的来说，这些研究表明 LACV Gc 与甲病毒 E1 和黄病毒 E 一样，具有 II 型融合蛋白的功能。 Gc 内有一个 22 个氨基酸的疏水片段 1066-1087，预计其在结构上与 SFV 和辛德比斯病毒 E1 的疏水结构域相关。该短序列在布尼亚病毒科中高度保守，并具有多个保守的半胱氨酸残基，其他 II 型蛋白（例如 SFV E1）也是如此。基于这些特征，在我们的诱变研究中，我们的工作假设是 LACV Gc (1066-1087) 充当其融合肽。在第一个具体目标中，我们将通过分析蚊子细胞中的融合并识别进一步将该区域与融合和进入相关联的肽和抗体来扩展这些研究。在第二个具体目标中，我们将使用新开发的反向遗传学系统，结合从分离的糖蛋白研究中获得的知识来构建 LACV 突变体。这些病毒将使我们能够将体外研究结果扩展到我们小组先前开发的 LACV 脑炎小鼠模型（第三个具体目标）。重要的是，由于该疏水区域在布尼亚病毒科中高度保守，因此该提案还将阐明其他新兴布尼亚病毒（包括 NIAID A 类和 C 类病原体 CCHFV 和 RVFV）之间的病毒融合和进入机制，并将对抗病毒治疗产生重大影响。公共卫生相关性拉克罗斯病毒是美国中西部地区小儿脑炎和无菌性脑膜炎的常见原因，该地区是其主要蚊媒三列蝽所在的地区。我们已经鉴定出拉克罗斯病毒糖蛋白 Gc 的融合肽。该提案中概述的研究将定义拉克罗斯病毒在哺乳动物和昆虫宿主中的融合和进入机制，确定新发现的融合结构域在 LACV 脑炎神经发病机制中的作用，并开发抗病毒疗法（融合肽）抑制剂和减毒病毒疫苗）。