Lazear Eric, Carfi Andrea, Whitbeck J Charles, Cairns Tina M, Krummenacher Claude, Cohen Gary H, Eisenberg Roselyn J
Department of Microbiology, University of Pennsylvania, 240 S. 40th St., Levy Rm. 233, Philadelphia, PA 19104, USA.
J Virol. 2008 Jan;82(2):700-9. doi: 10.1128/JVI.02192-07. Epub 2007 Nov 21.
Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.
糖蛋白D（gD）是单纯疱疹病毒（HSV）的受体结合蛋白，可与至少两种不同的蛋白受体——疱疹病毒进入介质（HVEM）和nectin-1结合。虽然两个受体结合区域都位于前234个氨基酸内，但晶体结构显示gD胞外域的C末端通常会遮挡受体结合位点。因此，受体结合必须取代C末端，并且推测这种构象变化是通过gB和gH/gL诱导融合所必需的。当在gD的第37位和第302位引入半胱氨酸残基时，会形成一个稳定C末端并阻止与任何一种受体结合的二硫键。我们推测，如果在更上游设计二硫键，受体结合可能会与融合诱导分离。为了验证这一点，我们又制作了另外五个双半胱氨酸突变体，每个突变体都可能在胞外域C末端和gD胞外域核心之间引入一个二硫键。预计施加最大限制的两个突变体无法结合受体或介导细胞间融合。然而，C末端最灵活的三个突变体与HVEM和nectin-1都结合良好。其中两个突变体在细胞间融合和空病毒互补方面受损。重要的是，该组中的第三个突变体在两种测定中均无功能。这个突变体清楚地将gD在触发融合中的作用与其在受体结合中的作用区分开来。基于这组突变体的特性，我们得出结论，与受体结合相比，融合需要gD胞外域C末端具有更大的灵活性。
