Envelope-receptor interactions in Nipah and Hendra virus pathobiology

描述(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(Nipah，NIV)和亨德拉(Hendra，HEV)病毒是副粘病毒科新定义的海尼帕病毒属成员。尼帕病毒(Nipah Virus，NIV)是一种紧急副粘病毒，可导致高达70%的感染患者发生致命性脑炎，越来越多的证据表明它在人与人之间传播。NIV在NIAID生物防御研究议程中被指定为优先病原体，如果用于打击养猪业，可能是农业生物恐怖主义的毁灭性因子。内皮合胞体是新城疫病毒感染的一种病原学特征，由融合(F)和附着(G)包膜糖蛋白介导。对新城疫病毒受体的鉴定将有助于阐明新城疫病毒感染的病理生物学机制，并促进有效治疗药物的合理开发。在我们的初步结果中，我们发现EphB类受体酪氨酸激酶(RTK)的膜结合配体ePhinB2与NIV的附着(G)糖蛋白特异性结合。可溶性Fc融合蛋白的ewitinB2有效地阻止了Niv的融合和进入，但不能有效地阻止ewitinB1。将ewitinB2导入非允许的细胞，使它们可以进行Niv融合和进入。表达于内皮细胞和神经元上，与已知的NIV的细胞趋向性一致。值得注意的是，NIV包膜介导的微血管内皮细胞和原代大脑皮层神经元的感染，可被可溶性ewitinB2抑制，但不能抑制相关的ewitinB1蛋白。综上所述，我们的数据表明ePhirinB2是NIV的一个功能性受体。我们还表明，相关蛋白ewitinB3可以作为另一种受体；ewitinB2和B3的不同用法可能解释了观察到的新城疫和戊型肝炎病毒之间的不同病原学特征。识别NIV受体为更全面地分析NIV包膜-受体相互作用打开了大门。我们提出以下具体目标，以增加我们对新城疫病理生物学的了解，并促进抗新城疫疫苗和治疗药物的发展。它们是：(1)鉴定与NIV-G和HEV-G相互作用的同源结构域和/或残基，(2)鉴定NIV-G和HEV-G中最小的受体结合域，并确定参与EPhin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ePhinB2相互作用的能力，以及(4)研究新型兔抗NIV-G单抗的性质。 公共卫生相关性：Nipah和Hendra病毒被指定为优先病原体，是致命的，可能是生物恐怖主义和农业恐怖主义的破坏性因素(对活畜业的破坏)。识别病毒受体使我们能够更好地研究病毒是如何进入细胞的。这些研究对于开发有效的抗Nipah疫苗和疗法至关重要。

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.