Stone Jacquelyn A, Vemulapati Bhadra M, Bradel-Tretheway Birgit, Aguilar Hector C
Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
Genomics and Proteomics Group, Department of Biotechnology, K L University, Greenfields, Guntur, Andhra Pradesh, India.
J Virol. 2016 Nov 14;90(23):10762-10773. doi: 10.1128/JVI.01469-16. Print 2016 Dec 1.
The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.
Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.
副粘病毒科包含许多具有重要医学意义的病毒，包括麻疹病毒、腮腺炎病毒、副流感病毒、呼吸道合胞病毒、人偏肺病毒，以及致命的人畜共患亨尼帕病毒——亨德拉病毒和尼帕病毒（NiV）。为了进入宿主细胞并在受感染宿主体内从一个细胞传播到另一个细胞，绝大多数副粘病毒利用两种病毒包膜糖蛋白：附着糖蛋白（G、H或血凝素神经氨酸酶[HN]）和融合糖蛋白（F）。G/H/HN与宿主细胞受体的结合会引发G/H/HN的结构变化，进而触发F经历一系列构象变化，导致病毒与细胞（病毒进入）或细胞与细胞（形成多核巨细胞）的膜融合。尽管一般认为副粘病毒的G/H/HN茎区与F头部区域相互作用，但在膜融合过程中G/H/HN和F实际相互作用的区域仍相对未知。确定此类相互作用区域的研究严重依赖于免疫共沉淀方法，其局限性包括使用去污剂以及蛋白质的胶束介导缔合。在此，我们开发了一种流式细胞术策略，通过交替使用全长形式的G和可溶性形式的F，或反之亦然，能够检测膜蛋白 - 蛋白相互作用。使用免疫共沉淀和流式细胞术策略，我们发现NiV G和F之间存在双齿相互作用，其中NiV G的茎区和头部区域均与F相互作用。这是副粘病毒的一项新的结构生物学发现。此外，我们的研究揭示了NiV G和F糖蛋白中对于G和F相互作用而言非必需的区域。
尼帕病毒（NiV）是一种人畜共患的副粘病毒，可导致人类高死亡率，目前尚无批准用于人类的治疗方法或疫苗。病毒进入宿主细胞依赖于两种病毒包膜糖蛋白：附着（G）糖蛋白和融合（F）糖蛋白。G与ephrinB2或ephrinB3细胞受体的结合会引发G的构象变化，进而导致F发生构象变化，从而导致病毒与宿主细胞膜融合及病毒进入。然而，目前尚不清楚在膜融合过程中G和F的哪些特定区域相互作用。过去确定相互作用区域的努力主要依赖于免疫共沉淀，这是一种存在一些缺陷的技术。我们开发了一种流式细胞术检测方法来研究膜蛋白 - 蛋白相互作用，并且使用该检测方法我们报告了一种双齿相互作用，即NiV G的头部和茎区均与NiV F相互作用，这是副粘病毒家族的一项新发现。
