Characterization of the La Crosse Virus glycoprotein fusion peptide

描述（由申请方提供）：拉克罗斯病毒（LACV）是一种NIAID B类优先病原体，是其主要蚊媒Ochlerotatus（原Aedes）trisariatus所在的美国中西部地区儿童脑炎和无菌性脑膜炎的常见病因。LACV基因组的结构组分（L、M和S）在病毒发病机制中具有基本的、明确的作用。我们实验室以前使用野生型LACV和TAHV 181/57（一种具有低神经侵袭性的高神经毒性毒株）的研究已经将神经侵袭表型映射到M片段，该片段编码Gn，Gc和非结构蛋白NSm。最近，使用重组糖蛋白，我们证明了对应于膜近端Gc的三分之二的区域，氨基酸860-1442，在介导融合和细胞进入中是至关重要的。进一步的计算分析鉴定了LACV Gc氨基酸区970-1350与两种甲病毒（辛德毕斯病毒和塞姆利基福雷斯特病毒（SFV））的E1融合蛋白之间的结构相似性。总的来说，这些研究表明LACV Gc，像甲病毒E1和黄病毒E一样，作为II型融合蛋白发挥作用。在Gc内，存在22个氨基酸的疏水区段1066-1087，其被预测为与SFV和辛德毕斯病毒E1的疏水结构域结构相关。短序列在布尼亚病毒科中高度保守，并具有几个保守的半胱氨酸残基，其他II型蛋白如SFV E1也是如此。基于这些特征，并且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。在第一个具体目标中，我们将通过分析蚊子细胞中的融合来扩展这些研究，并通过鉴定进一步将该区域与融合和进入相关联的肽和抗体。在第二个具体的目标，我们将使用一个新开发的反向遗传学系统，构建LACV突变体纳入从分离的糖蛋白的研究中获得的知识。这些病毒将使我们能够将我们的体外发现扩展到我们小组先前开发的LACV脑炎小鼠模型（第三个具体目标）。重要的是，由于该疏水性区域在布尼亚病毒科中高度保守，因此该提议还将阐明其他新出现的布尼亚病毒（包括NIAID A类和C类病原体CCHFV和RVFV）之间的病毒融合和进入机制，并将对抗病毒治疗产生重大影响。公共卫生相关性拉克罗斯病毒是美国中西部地区儿童脑炎和无菌性脑膜炎的常见病因，其主要蚊媒三列黄蚊（Ochlerotatus triseriatus）居住在该地区。我们鉴定了拉克罗斯病毒糖蛋白Gc的融合肽。本提案中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，确定新鉴定的融合结构域在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).