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: 8274784
• 负责人: Samantha Standish Soldan
• 金额: $ 34.21万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8274784
• 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; 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），NIAID类别B优先病原体，是感染的常见原因。 美国中西部地区的小儿脑炎和无菌性脑膜炎 其主要蚊媒Ochlerotatus（以前称为Aedes）trisariatus居住的地方。 LACV基因组的结构组分（L、M和S）具有基本的、良好的- 在病毒发病机制中的作用。我们实验室以前的研究使用野生型 LACV和TAHV 181/57是一种高度神经毒力的低神经侵袭性毒株， 将神经侵袭性表型映射到M片段，其编码Gn、Gc和a 非结构蛋白NSm。最近，利用重组糖蛋白， 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关键的。进一步 计算分析鉴定了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，像 甲病毒E1和黄病毒E作为II型融合蛋白发挥功能。在GC中， 是一个22个氨基酸的疏水片段，1066-1087，预测其与 在结构上具有SFV和辛德毕斯病毒E1的疏水结构域。短 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，如其他II型蛋白，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）作为其融合肽发挥功能。 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，并通过鉴定肽和抗体，进一步将该区域与 融合和进入。在第二个具体目标，我们将使用一个新开发的反向 构建LACV突变体的遗传学系统， 分离糖蛋白的研究。这些病毒会让我们 我们先前开发的LACV脑炎小鼠模型的体外研究结果 第三个目标（第三个具体目标）。重要的是，由于该疏水区域高度保守 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，并将对抗病毒治疗具有重要意义。拉克罗斯病毒是儿童脑炎和无菌性脑膜炎的常见病因， 美国中西部，主要的蚊子媒介，三列黄蚊 居住。我们鉴定了拉克罗斯病毒糖蛋白Gc的融合肽。 这项建议中概述的研究将确定La的融合和进入机制 交叉病毒在哺乳动物和昆虫宿主中的作用，决定了新的 在LACV脑炎的神经发病机制中确定了融合结构域，并开发了 抗病毒疗法（融合肽抑制剂和减毒病毒疫苗）。

项目成果

La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic.

拉克罗斯病毒 (LACV) Gc 融合肽突变体的生长和融合表型受损，但仍然具有神经毒性。

• DOI: 10.1016/j.virol.2010.04.012
• 发表时间: 2010
• 期刊: Virology
• 影响因子: 3.7
• 作者: Soldan,SamanthaS;Hollidge,BradleyS;Wagner,Valentina;Weber,Friedemann;Gonzalez-Scarano,Francisco
• 通讯作者: Gonzalez-Scarano,Francisco

Chronic viral infection and primary central nervous system malignancy.

• DOI: 10.1007/s11481-010-9204-0
• 发表时间: 2010-09
• 期刊: JOURNAL OF NEUROIMMUNE PHARMACOLOGY
• 影响因子: 6.2
• 作者: Saddawi-Konefka, Robert;Crawford, John R.
• 通讯作者: Crawford, John R.

Viral infections of the central nervous system: pathogenesis to therapeutics.

中枢神经系统的病毒感染：发病机制到治疗。

• DOI: 10.1007/s11481-010-9231-x
• 发表时间: 2010
• 期刊: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology
• 作者: Soldan,SamanthaS;Jacobson,Steven
• 通讯作者: Jacobson,Steven

The role of interferon antagonist, non-structural proteins in the pathogenesis and emergence of arboviruses.

• DOI: 10.3390/v3060629
• 发表时间: 2011-06
• 期刊: Viruses
• 作者: Hollidge BS;Weiss SR;Soldan SS
• 通讯作者: Soldan SS