Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L
Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L
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DOI：
10.1128/jvi.00061-17
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发表时间：
2017-05-01
影响因子：
5.4
通讯作者：
Fuchs, Walter
中科院分区：
文献类型：
--
作者：
Vallbracht, Melina;Rehwaldt, Sascha;Fuchs, Walter
几种包膜糖蛋白参与疱疹病毒进入细胞、直接细胞间传播和诱导细胞融合。膜融合蛋白糖蛋白B（gB）和可能的gB激活异源二聚体gH/gL是这些过程所必需的，并在整个疱疹病毒科中保守。然而，在α疱疹病毒伪狂犬病病毒（PrV）的gL阴性突变体的延长细胞培养传代后，可以分离出表型回复突变体，其获得了影响gH胞外域的gL相互作用N末端部分（gDH和gH（B4.1））的自发突变（B. G. Klupp和T. C. Mettenleiter，J Virol 73：30143022，1999; C. Schroter，M. Vallbracht，J. Altenschalet，S. Kargoll，W.福克斯，B。G. Klupp和T. C. Mettenleiter，J Virol 90：2264 - 2272，2016）。为了更详细地研究gH的这一部分的功能相关性，我们在质粒克隆的gH基因（gH（32/98））的5'端引入了66个密码子的框内缺失。保留N-末端信号肽，并且缺失不影响gH的表达或加工，但在体外融合测定中废除其功能。将工程gH基因插入PrV基因组导致缺陷突变体（pPrV-gH（32/98）K），其不能进入和传播。有趣的是，当突变gH（32/98）与从PrV传代gL缺失突变体中获得的超致敏gB（B4.1）共表达时，突变gH（32/98）的体外活性得到恢复。此外，pPrV-gH（32/98）K的进入和扩散缺陷被gB（B4.1）中的顺式和反式突变所补偿，而不依赖于gL。因此，PrV gL和gH的gL相互作用结构域不是功能的严格要求。重要性膜融合对于包膜病毒的感染性进入和传播至关重要。虽然许多包膜病毒仅需要一种或两种蛋白质用于受体结合和膜融合，但疱疹病毒感染依赖于几种包膜糖蛋白。除了亚家族特异性受体结合蛋白，核心融合机制由保守的融合蛋白gB和gH/gL复合物组成。后者的作用尚不清楚，但假设与gB相互作用以进行融合激活。使用同基因病毒重组体，我们在这里证明，gL和gL-结合域的PrV gH的膜融合不严格需要在病毒进入和传播时，伴随的突变gB的存在，增加其融合性。因此，我们的研究结果强烈支持融合过程中的功能gB-gH相互作用的概念。
Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV), phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH and gH(B4.1)) (B. G. Klupp and T. C. Mettenleiter, J Virol 73: 30143022, 1999; C. Schroter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J Virol 90: 2264 -2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5' end of the plasmid-cloned gH gene (gH(32/98)). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH(32/98)K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH(32/98) was restored when it was coexpressed with hyperfusogenic gB(B4.1), obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH(32/98)K were compensated by the mutations in gB(B4.1) in cis, as well as in trans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function.IMPORTANCE Membrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear, but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants, we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process.