Aguilar Hector C, Ataman Zeynep Akyol, Aspericueta Vanessa, Fang Angela Q, Stroud Matthew, Negrete Oscar A, Kammerer Richard A, Lee Benhur
Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
J Biol Chem. 2009 Jan 16;284(3):1628-35. doi: 10.1074/jbc.M807469200. Epub 2008 Nov 19.
Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (beta6S4/beta1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.
副粘病毒进入细胞需要附着(G/H/HN)糖蛋白和融合(F)糖蛋白的协同作用,但在病毒进入过程中,受体结合如何激活G以触发F介导的融合尚不清楚。在这里,我们在尼帕病毒(NiV)G中鉴定出一个受体(ephrinB2)诱导的变构激活位点,该位点参与触发F介导的融合。我们首先生成了一种构象单克隆抗体(单克隆抗体45(Mab45)),其与NiV-G的结合在NiV-G与ephrinB2结合后增强。然而,Mab45也抑制病毒进入,并且其受体结合增强(RBE)表位是温度依赖性的,这表明G上的Mab45 RBE表位可能参与触发F。Mab45 RBE表位被定位到球状结构域的基部(β6S4/β1H1)。该区域内未表现出这种RBE表位的丙氨酸扫描突变体尽管能够结合ephrinB2、寡聚化并以野生型(WT)水平与F结合,但也不具有融合能力。尽管圆二色性显示在添加ephrinB2后WT NiV-G的可溶性胞外域发生了构象变化,但可溶性RBE表位突变体或短柄G突变体未检测到此类变化。此外,WT G而非RBE表位突变体在ephrinB2结合后可与F解离。最后,使用生物素化的HR2肽检测前发夹中间体的形成(F触发的一个主要特征),我们表明ephrinB2与WT G结合而非与RBE表位突变体结合可触发F。总之,我们认为NiV-G球状头部结构域基部与柄部结构域之间的协同相互作用在介导病毒进入过程中受体诱导的F触发中起作用。