Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site
Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site
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DOI：
10.1128/jvi.01255-17
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发表时间：
2017-12-01
影响因子：
5.4
通讯作者：
Longnecker, Richard
中科院分区：
文献类型：
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作者：
Mohl, Britta S.;Chen, Jia;Longnecker, Richard
Epstein-Barr病毒（EBV）进入上皮细胞是由保守的核心融合机制介导的，所述核心融合机制由融合原gB和受体结合复合物gH/gL组成。异二聚体gH/gL复合物结合EBV上皮细胞受体或gp 42，其结合B细胞受体，触发gB介导的病毒体包膜与细胞膜的融合。我们的前期研究发现，gL糖基化突变体N69 L/S71 V具有上皮细胞特异性超流表型。为了研究这种gL突变体对gB驱动的上皮细胞融合的起始和动力学的影响，我们建立了一种无病毒的分裂绿色荧光蛋白细胞-细胞融合测定，其能够使用活细胞实时测量膜融合。gL_N69L/S71 V突变体在早期时间点开始具有比野生型gL高多达300%的上皮细胞融合活性的大幅增加。超灌流性的gL突变体是不是一个结果的改变，在复杂的形成与gH或改变细胞定位。此外，gL突变体的hypperfusogenic表型与扩大合胞体的形成相关。总之，我们目前的研究结果突出了gL在gB介导的上皮细胞融合的动力学中的重要作用，增加了以前的研究结果表明gL和gB之间的直接相互作用在EBV膜fusion.IMPORTANCE EBV主要感染上皮细胞和B淋巴细胞，这是EBV相关恶性肿瘤霍奇金淋巴瘤和伯基特淋巴瘤以及鼻咽癌的起源细胞。与核心融合机制的其他关键参与者相反，gL在EBV诱导的膜融合过程中具有最难以捉摸的作用。我们发现gL的糖基化位点N69/S71参与限制上皮细胞融合活性，与合胞体大小密切相关。有趣的是，我们的数据显示，gL糖基化突变体增加了超促流gB突变体的融合活性，表明该gL突变体和gB突变体靶向融合过程中的不同步骤。我们关于gL和gB如何共同调节上皮细胞融合动力学的研究对于了解EB病毒对上皮细胞和B淋巴细胞的高度调节的嗜性至关重要，并且可能会产生防止病毒进入靶宿主细胞的治疗新策略。最后，使我们的结果特别感兴趣的是在其他疱疹病毒gL合胞体突变体的情况下。
Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.