Envelope-receptor interactions in Nipah and Hendra virus pathobiology

描述（由申请人提供）：新出现的病毒病原体对美国的健康和经济构成严重威胁。尼帕（NiV）和亨德拉（HeV）病毒是副粘病毒科的新定义的亨帕病毒属的成员。尼帕病毒（NiV）是一种新出现的副粘病毒，在高达70%的感染患者中引起致命性脑炎，并且越来越多的证据表明人传人。NiV在NIAID生物防御研究议程中被指定为优先病原体，如果用于养猪业，可能成为农业生物恐怖主义的破坏性因子。内皮合胞体是NiV感染的特征性特征，并且由融合（F）和附着（G）包膜糖蛋白介导。NiV受体的鉴定将揭示NiV感染的病理生物学，并刺激有效治疗方法的合理开发。在我们的初步结果中，我们表明ephrinB 2，ephB类受体酪氨酸激酶（RTK）的膜结合配体，特异性结合到NiV的附着（G）糖蛋白。ephrinB 2而不是ephrinB 1的可溶性Fc融合蛋白有效地阻断了NiV融合和进入。将ephrinB 2转染到非允许细胞中使得它们允许NiV融合和进入。EphrinB 2在内皮细胞和神经元上表达，与NiV的已知细胞向性一致。值得注意的是，NiV包膜介导的微血管内皮细胞和原代皮层大鼠神经元的感染，被抑制可溶性ephrinB 2，但不相关ephrinB 1蛋白。累积起来，我们的数据表明ephrinB 2是NiV的功能性受体。我们还表明，ephrinB 3，一种相关的蛋白质，可以作为一种替代受体; ephrinB 2与B3的差异使用可以解释NiV和HeV之间观察到的变异致病谱。识别NiV受体为更全面地分析NiV受体-受体相互作用打开了大门。我们提出以下具体目标，以增加我们对NiV病理生物学的理解，并促进抗NiV疫苗和治疗剂的开发。它们是：（1）鉴定肝配蛋白B2/B3中介导与NiV-G和HeV-G相互作用的同源结构域和/或残基，（2）表征NiV-G和HeV-G中的最小受体结合结构域，并鉴定参与肝配蛋白受体相互作用的关键残基，（3）开发和确定小分子拮抗剂阻断NiV-G与肝配蛋白B2相互作用的能力，（4）研究新型抗NiV-G单克隆抗体的特性。 公共卫生相关性：尼帕病毒和亨德拉病毒被指定为优先病原体，是致命的，并且可以是生物恐怖主义和农业恐怖主义的破坏性因子（对畜牧业的破坏）。识别病毒受体使我们能够更好地研究病毒如何进入细胞。这些研究对于开发有效的抗尼帕疫苗和治疗方法至关重要。

DESCRIPTION (provided by applicant): Emerging viral pathogens present a critical threat to U.S. health and economy. Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytia is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the NiV receptor will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. In our preliminary results, we show that ephrinB2, the membrane bound ligand for the ephB class of receptor tyrosine kinases (RTKs), specifically bound to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2 but not ephrinB1 effectively blocked NiV fusion and entry. Transfection of ephrinB2 into non-permissive cells rendered them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. Significantly, NiV envelope mediated infection of microvascular endothelial cells, and primary cortical rat neurons, was inhibited by soluble ephrinB2, but not the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV. We also show that ephrinB3, a related protein, can serve as an alternative receptor; differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Identifying the NiV receptor opens the door for a more comprehensive analysis of the NiV envelope-receptor interactions. We propose the following Specific Aims to increase our understanding of NiV pathobiology, and facilitate the development of anti-NiV vaccines and therapeutics. They are: (1) Identify cognate domains and/or residues in ephrinB2/B3 that mediate interactions with NiV-G and HeV-G, (2) Characterize the minimal receptor binding domain in NiV-G and HeV-G, and identify the critical residues involved in ephrin receptor interactions, (3) Develop and define the ability of small molecule antagonists to block NiV-G's interaction with ephrinB2, and (4) Investigate the properties of novel rabbit monoclonal antibodies against NiV-G. Public Health Relevance: Nipah and Hendra viruses are designated priority pathogens, are deadly, and can be devastating agents of bioterrorism and agroterrorism (devastation of the live-stock industry). Identifying the virus receptor allows us to better study how the virus gets into cells. These investigations are crucial to the development of effective anti-Nipah vaccines and therapeutics.