Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions
Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions
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DOI：
10.1128/jvi.02710-06
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发表时间：
2007-04-01
影响因子：
5.4
通讯作者：
Cohen, Gary H.
中科院分区：
文献类型：
--
作者：
Bender, Florent C.;Samanta, Minu;Cohen, Gary H.
糖蛋白B（gB），沿着gD、gH和gL，是单纯疱疹病毒（HSV）侵入所必需的。gB胞外域的晶体结构显示它是延长的多结构域三聚体。我们产生并表征了一组67种单克隆抗体（MAbs）。11个单克隆抗体具有病毒中和活性。为了将gB组织到这些结构域内的功能区域中，我们定位了由整个单克隆抗体组识别的表位，并将其映射到gB的晶体结构上。大多数单克隆抗体针对连续或不连续表位，但有几种识别的不连续表位显示出一定的变性抗性，我们将其称为假连续表位。每个类别都含有一些具有中和活性的单克隆抗体。为了绘制连续表位，我们使用了跨越gB胞外域的重叠肽，并通过酶联免疫吸附测定法测量结合。为了鉴定不连续和假连续表位，将gB的胞外域的纯化形式gB（730 t）通过α-胰凝乳蛋白酶切割成两个主要片段，其包含氨基酸98至472（结构域I和II）和氨基酸473至730（结构域III、IV和V的主要部分）。我们还构建了一系列的gB截断，以增加其他映射策略。最后，我们使用生物传感器分析将单克隆抗体分配到竞争组。总之，我们的结果确定了四个功能区：（i）一个由结构域I内的残基和结构域V的氨基酸697至725形成;（ii）第二个由残基391至410、残基454至475和结构域11内的不太确定的区域形成;（iii）包含结构域IV的残基的区域，其靠近结构域III;和（iv）在晶体结构中未分辨的N末端的前12个残基。我们的数据表明，多个域的gB功能是至关重要的。
Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain 11; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.