Characterization of the La Crosse Virus glycoprotein fusion peptide

描述(由申请人提供)：La Crosse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，美国中西部地区是其主要蚊媒Ochlerotatus(前三日伊蚊)居住的地区。LACV基因组的结构成分(L、M和S)在病毒致病过程中具有基本的、明确的作用。我们实验室以前使用野生型LACV和TAHV 181/57株的研究已经将神经侵袭性表型定位到编码Gn、GC和非结构蛋白NSM的M节段。最近，我们使用重组糖蛋白证明了对应于GC膜近三分之二的氨基酸860-1442的区域在介导融合和细胞进入方面是关键的。进一步的计算分析发现，LACV的GC氨基酸序列970-1350与两种甲型病毒：辛德比斯病毒和塞姆利基福雷斯特病毒(SFV)的E1融合蛋白结构相似。总而言之，这些研究表明，LACV GC与甲型病毒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22个氨基酸的疏水片段，1066-1087，被预测与SFV和Sindbis病毒E1的疏水结构域在结构上相关。该短序列在布尼亚病毒科中高度保守，具有几个保守的半胱氨酸残基，与其他类型II蛋白一样，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)作为其融合肽发挥作用。在第一个具体目标中，我们将通过分析蚊子细胞中的融合，并通过识别进一步将该区域与融合和进入联系起来的多肽和抗体来扩展这些研究。在第二个具体目标中，我们将利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。然后，这些病毒将允许我们将体外发现扩展到我们小组之前开发的LACV脑炎小鼠模型(第三个特定目标)。重要的是，由于这个疏水区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出现的布尼亚病毒之间的融合和进入机制，包括NIAID A和C类病原体CCHFV和RVFV，并将对抗病毒治疗具有重要意义。与公共卫生相关的La Crosse病毒是美国中西部儿童脑炎和无菌性脑膜炎的常见原因，它的主要蚊媒Ochlerotatus triseriatus居住在那里。我们已经鉴定了La Crosse病毒糖蛋白GC的融合肽。这项建议中概述的研究将确定La Crosse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)。

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).