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: 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
• 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

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，是美国中西部地区儿童脑炎和无菌性脑膜炎的常见原因，其主要蚊子载体是三翼伊蚊（Ochlerotatus，以前称为伊蚊）。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的、明确的作用。我们实验室先前使用野生型LACV和TAHV 181/57（一种具有低神经侵袭性的高神经毒性菌株）进行的研究已经将神经侵袭表型定位到编码Gn， Gc和非结构蛋白NSm的M段。最近，利用重组糖蛋白，我们证明了与Gc近三分之二的膜相对应的区域，氨基酸860-1442，在介导融合和细胞进入中至关重要。进一步的计算分析确定了两种甲型病毒：Sindbis病毒和Semliki Forrest病毒（SFV）的LACV Gc氨基酸区970-1350与E1融合蛋白的结构相似性。总之，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。在Gc中有一个由22个氨基酸组成的疏水片段1066-1087，被预测在结构上与SFV和Sindbis病毒E1的疏水结构域相关。这个短序列在布尼亚病毒科中高度保守，与其他II型蛋白（如SFV E1）一样，具有几个保守的半胱氨酸残基。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）作为其融合肽起作用。在第一个特定目标中，我们将通过分析蚊子细胞中的融合，并通过识别进一步将该区域与融合和进入联系起来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来构建LACV突变体，并结合从分离的糖蛋白研究中获得的知识。然后，这些病毒将使我们能够将我们的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（第三个特定目标）。重要的是，由于该疏水区域在布尼亚病毒科中高度保守，这一建议也将阐明病毒在其他新兴布尼亚病毒（包括NIAID A类和C类病原体CCHFV和RVFV）中的融合和进入机制，并将对抗病毒治疗产生重大影响。拉克罗斯病毒是美国中西部儿童脑炎和无菌性脑膜炎的常见病因，它的主要蚊子载体是三爪蟾。我们已经确定了拉克罗斯病毒糖蛋白Gc的融合肽。本提案概述的研究将确定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，并开发抗病毒治疗方法（融合肽抑制剂和减毒病毒疫苗）。