Characterization of the La Crosse Virus glycoprotein fusion peptide

La Crosse病毒(LACV)是NIAID B类优先病原体，是引起 美国中西部地区的儿童脑炎和无菌性脑膜炎 它的主要蚊媒Ochlerotatus(前身为伊蚊)Tisariatus就居住在那里。 LACV基因组的结构成分(L、M和S)具有基本的、良好的- 明确了病毒发病机制中的角色。我们实验室以前使用野生型的研究 LACV和TAHV 181/57是一种高神经毒力、低神经侵袭性的毒株，具有 将神经侵袭性表型映射到编码Gn、GC和a的M片段 非结构蛋白，NSM。最近，我们使用重组糖蛋白 显示与GC近三分之二的膜相对应的区域， 氨基酸860-1442在调节融合和细胞进入中起关键作用。进一步 计算分析确定了LACV GC氨基酸的结构相似性 辛德比斯病毒和塞姆利奇病毒的970-1350区和E1融合蛋白 福雷斯特病毒(SFV)。总而言之，这些研究表明，LACV GC与 甲型病毒E1和黄病毒E的功能类似于II型融合蛋白。在GC内部， 是一个22个氨基酸的疏水片段，1066-1087，预测与 在结构上与SFV和辛德比斯病毒E1的疏水结构域同源。短小的 序列在布尼亚病毒科中高度保守，并具有几个特征 保守的半胱氨酸残基，以及其他类型II蛋白，如SFV E1。基于 这些特征，以及在我们的突变研究中，我们的工作假设是LACV GC(1066-1087)是其融合肽。 在第一个具体目标中，我们将通过分析蚊子中的融合来扩展这些研究 细胞，并通过鉴定与该区域进一步相关的多肽和抗体 融合和进入。在第二个具体目标中，我们将使用新开发的反转 构建LACV突变体的遗传学系统 分离的糖蛋白的研究。然后，这些病毒将允许我们延长我们的 我们先前建立的LACV脑炎小鼠模型的体外实验结果 组(第三个具体目标)。重要的是，由于这一疏水区域高度保守 在布尼亚病毒科中，这一建议也将阐明病毒融合的机制 和进入包括NIAID A和C类在内的其他新出现的布尼亚病毒 病原体CCHFV和RVFV，并将对抗病毒治疗具有重大意义。拉克罗斯病毒是引起儿童脑炎和无菌性脑膜炎的常见原因。 美国中西部，在那里它的主要蚊媒，Ochlerotatus triseriatus 住在那里。我们已经鉴定了La Crosse病毒糖蛋白GC的融合肽。 本提案中概述的研究将确定La的融合和进入机制 Crosse病毒在哺乳动物和昆虫中的寄主，确定了新的作用 确定LACV脑炎神经发病机制中的融合结构域，并发展 抗病毒疗法(融合肽抑制剂和减毒病毒疫苗)。

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).