Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R
Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R
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DOI：
10.1128/jvi.79.10.6260-6271.2005
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发表时间：
2005-05-01
影响因子：
5.4
通讯作者：
Cohen, GH
中科院分区：
文献类型：
--
作者：
Aldaz-Carroll, L;Whitbeck, JC;Cohen, GH
痘苗病毒胞外被膜病毒(EEV)是病毒在体外和体内细胞间和远距离传播的关键。B5R基因编码一种EEV特异性的I型膜蛋白，它是有效形成EEV所必需的。B5R的大部分胞外结构域由四个结构域组成，与短共识重复结构域同源，紧随其后的是茎。以往的研究表明，针对B5R胞外区的多克隆抗体可抑制EEV感染。在这项研究中，我们的目标是阐明B5R的抗原结构并将其与其功能联系起来。为此，我们使用杆状病毒表达系统生产了多毫克数量的痘苗病毒B5R作为可溶性蛋白[B5R(275T)]。然后我们选择并鉴定了一组识别B5R(275T)的26个单抗(MAb)。其中5个单抗中和了EEV并抑制了彗星的形成。另外两个单抗只能中和EEV，而另外五个只能抑制彗星的形成。这表明EEV中和试验和彗星抑制试验测量了不同的病毒功能，B5R上至少有两个不同的抗原部位对这些活性是重要的。我们通过肽图谱和生物传感器分析的相互单抗阻断研究，进一步表征了B5R(275T)的单抗和抗原结构。中和单抗识别的表位定位于SCR1-SCR2和/或B5R(275T)的茎上。此外，肽和阻断数据支持这样的概念，即SCR1和茎可能是并列的，并且可能是同一功能域的一部分。
Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.