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: 7880387
• 负责人: Francisco Gonzalez-Scarano
• 金额: $ 2.11万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-14 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880387
• 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; therapeutic development; vector mosquito; virus pathogenesis

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 previouly 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. 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).

拉克罗斯病毒 (LACV) 是 NIAID B 类优先病原体，是导致以下疾病的常见原因： 美国中西部地区的小儿脑炎和无菌性脑膜炎 其主要蚊媒三色蚊（Ochlerotatus，以前称为伊蚊）居住于此。 LACV 基因组的结构成分（L、M 和 S）具有重要的、良好的- 在病毒发病机制中的明确作用。我们实验室之前使用野生型进行的研究 LACV 和 TAHV 181/57 是一种具有低神经侵袭性的高神经毒株， 将神经侵袭表型映射到 M 片段，编码 Gn、Gc 和 a 非结构蛋白，NSm。最近，我们利用重组糖蛋白 证明对应于膜近端 Gc 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。更远 计算分析确定了 LACV Gc 氨基酸之间的结构相似性 区域 970-1350 和两种甲病毒的 E1 融合蛋白：辛德毕斯病毒和塞姆利基病毒 福雷斯特病毒（SFV）。总的来说，这些研究表明 LACV Gc，就像 甲病毒E1和黄病毒E，作为II型融合蛋白发挥作用。 GC内有 是一个 22 个氨基酸的疏水片段，1066-1087，预计与 结构上具有 SFV 和 Sindbis 病毒 E1 的疏水结构域。短的 该序列在布尼亚病毒科内高度保守，并具有几个特点： 保守的半胱氨酸残基，其他 II 型蛋白也是如此，例如 SFV E1。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的融合来扩展这些研究 细胞，并通过识别进一步将该区域与 融合和进入。在第二个具体目标中，我们将使用新开发的反向 结合从获得的知识构建 LACV 突变体的遗传学系统 分离糖蛋白的研究。这些病毒将使我们能够扩展我们的研究范围 我们之前开发的 LACV 脑炎小鼠模型的体外研究结果 组（第三个具体目标）。重要的是，由于这个疏水区域是高度保守的 在布尼亚病毒科中，该提案还将阐明病毒融合机制 以及其他新兴布尼亚病毒（包括 NIAID A 类和 C 类）的进入 病原体 CCHFV 和 RVFV，将对抗病毒治疗产生重大影响。拉克罗斯病毒是儿童脑炎和无菌性脑膜炎的常见原因 美国中西部，主要蚊媒三列蝽 居住。我们已经鉴定出拉克罗斯病毒糖蛋白 Gc 的融合肽。 本提案中概述的研究将定义 La 的融合和进入机制 Crosse 病毒在哺乳动物和昆虫宿主中，确定新的作用 确定了 LACV 脑炎神经发病​​机制中的融合结构域，并开发 抗病毒疗法（融合肽抑制剂和减毒病毒疫苗）。