Characterization of the La Crosse Virus glycoprotein fusion peptide

拉克罗斯病毒糖蛋白融合肽的表征

• 批准号: 7878107
• 负责人: Francisco Gonzalez-Scarano
• 金额: $ 34.55万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/7878107
• 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

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）：在美国中西部的主要蚊子媒介，Ochlerotatus（曾是艾德斯）Trisariatus，Resides，Resides，Resix naiD类病毒（LACV）是一种NIAID类别的B PRIPITY病原体，是儿童脑炎和无菌脑膜炎的常见原因。 LACV基因组（L，M和S）的结构成分在病毒发病机理中具有必不可少的定义作用。我们实验室使用野生型LACV和TAHV 181/57的先前研究是一种高度神经毒性菌株，具有低神经性侵袭性，已将神经脱落的表型映射到编码GN，GC和非结构蛋白NSM，NSM，NSM，NSM，NSM。最近，使用重组糖蛋白，我们证明了对应于GC膜近端三分之二的区域，即氨基酸860-1442，对于介导融合和细胞的进入至关重要。进一步的计算分析确定了LACV GC氨基酸区域970-1350与两种α病毒的E1融合蛋白之间的结构相似性：Sindbis病毒和Semliki Forrest病毒（SFV）。总的来说，这些研究表明，LACV GC（如Alphavirus E1和Flavivirus E）起作用，是II型融合蛋白。在GC中，有一个22个氨基酸疏水片段1066-1087，预计将与SFV和Sindbis病毒E1的疏水结构域结构相关。短序列在Bunyaviridae家族中高度保守，并具有几种保守的半胱氨酸残基，其他II型蛋白（例如SFV E1）也是如此。基于这些特征和我们的诱变研究，我们的工作假设是LACV GC（1066-1087）用作其融合肽。在第一个具体目的中，我们将通过分析蚊子细胞中的融合以及鉴定将该区域与融合和进入的肽和抗体进行分析，扩展这些研究。在第二个特定目的中，我们将使用新开发的反向遗传系统来构建从分离的糖蛋白研究中获得的知识的LACV突变体。然后，这些病毒将使我们能够将我们的体外发现扩展到以前由我们组开发的LACV脑炎的小鼠模型（第三个特定目的）。重要的是，由于该疏水区在Bunyaviridae中是高度保守的，因此该提案还将阐明病毒融合的机制和其他新兴的Bunyaviruse的机制，包括NIAID类别A和C类病原体CCHFV和RVFV，并将对抗病毒治疗产生重大影响。公共卫生相关性La Crosse病毒是美国中西部的小儿脑炎和无菌性脑膜炎的常见原因，在该州主要蚊子媒介（Ochlerotatus triseriatus）居住。我们已经确定了La Crosse病毒糖蛋白GC的融合肽。该提案中概述的研究将定义哺乳动物和昆虫宿主中La Crosse病毒的融合机制，并确定新鉴定的融合结构域在LACV脑炎的神经发生中的作用，并开发抗病毒疗法（融合肽抑制剂和促肽）。