Vallbracht Melina, Rehwaldt Sascha, Klupp Barbara G, Mettenleiter Thomas C, Fuchs Walter
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
J Virol. 2017 Apr 13;91(9). doi: 10.1128/JVI.00061-17. Print 2017 May 1.
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 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) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014-3022, 1999; C. Schröter, 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). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gHK), which was incapable of entry and spread. Interestingly, activity of mutated gH was restored when it was coexpressed with hyperfusogenic gB, obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gHK were compensated by the mutations in gB in , as well as in , independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function. 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.
几种包膜糖蛋白参与疱疹病毒进入细胞、细胞间直接传播以及细胞融合的诱导。膜融合蛋白糖蛋白B（gB）以及推测的gB激活异二聚体gH/gL对于这些过程至关重要，并且在整个疱疹病毒亚科中都保守。然而，在α疱疹病毒伪狂犬病病毒（PrV）的gL阴性突变体进行长时间细胞培养传代后，可以分离出表型回复突变体，这些突变体获得了影响gH胞外域（gDH和gH）与gL相互作用的N端部分的自发突变（B.G. Klupp和T.C. Mettenleiter，《病毒学杂志》73:3014 - 3022，1999；C. Schröter、M. Vallbracht、J. Altenschmidt、S. Kargoll、W. Fuchs、B.G. Klupp和T.C. Mettenleiter，《病毒学杂志》90:2264 - 2272，2016）。为了更详细地研究gH这一部分的功能相关性，我们在质粒克隆的gH基因（gH）的5'端引入了66个密码子的框内缺失。保留了N端信号肽，该缺失不影响gH的表达或加工，但在融合试验中废除了其功能。将工程化的gH基因插入PrV基因组产生了一个缺陷突变体（pPrV - gHK），它无法进入细胞和传播。有趣的是，当与从PrV的传代gL缺失突变体获得的高融合性gB共表达时，突变gH的活性得以恢复。此外，pPrV - gHK的进入和传播缺陷在PrV的gB突变体以及gB突变体中得到补偿，与gL无关。因此，PrV gL和gH的gL相互作用域对于功能并非严格必需。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进行受体结合和膜融合，但疱疹病毒感染依赖于几种包膜糖蛋白。除了亚科特异性受体结合蛋白外，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，但据推测它与gB相互作用以激活融合。使用同基因病毒重组体，我们在此证明，当同时存在增加其融合性的gB突变时，PrV gL和PrV gH的gL结合域在病毒进入和传播过程中对于膜融合并非严格必需。因此，我们的结果有力地支持了融合过程中功能性gB - gH相互作用的观点。
