Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein
Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein
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DOI：
10.1016/j.virol.2006.04.041
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发表时间：
2006-09-01
期刊：
影响因子：
3.7
通讯作者：
Blissard, Gary W.
中科院分区：
文献类型：
--
作者：
Zhou, Jian;Blissard, Gary W.
褐斑潜蝇核型多角体病毒(AcMNPV)囊膜糖蛋白GP64是病毒侵入过程中芽胞病毒(BV)囊膜与囊膜融合所需的酸诱导膜融合活性的充分必要条件。出芽病毒(BV)的传染性被针对GP64的单抗AcV1中和。先前的研究表明，AcV1识别构象表位，并不抑制病毒与细胞的附着，而是在病毒附着之后的某个步骤抑制病毒进入。我们发现，当GP64暴露在低pH(pH 4.5)中时，GP64的AcV1识别会丢失，并通过将GP64恢复到pH 6.2来恢复。此外，GP64在十二烷基硫酸钠中变性后，AcV1表位丢失，但在没有十二烷基硫酸钠的情况下，通过重折叠可以恢复AcV1表位。利用在昆虫细胞中表达的截短的GP64蛋白，我们将AcV1表位定位到GP64中央可变区的24个氨基酸区域。当映射的AcV1表位内的序列被c-Myc表位取代时，所得到的构建物被用来取代重组AcMNPV病毒中的wt GP64，修饰的GP64蛋白似乎具有正常的功能。然而，抗cMyc的单抗并不能中和这些病毒的传染性。由于c-Myc单抗与GP64序列中相同位置的结合没有导致中和，这些研究表明AcV1中和可能是由于AcV1结合引起的特定结构限制，而不仅仅是由于GP64中该位置抗体结合引起的空间位阻。(C)2006 Elsevier Inc.保留所有权利。
The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the enclosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function nortnally. However, an anti-cMyc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64. (c) 2006 Elsevier Inc. All rights reserved.