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Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site
Journal of Virology ( IF 5.4 ) Pub Date : 2017-12-01 , DOI: 10.1128/jvi.01255-17
Britta S Möhl 1 , Jia Chen 1 , Seo Jin Park 1 , Theodore S Jardetzky 2 , Richard Longnecker 3
Affiliation  

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.



中文翻译:

gB 触发的 Epstein-Barr 病毒与上皮细胞的融合受到 gL 糖基化位点的限制

EB 病毒 (EBV) 进入上皮细胞是由保守的核心融合机制介导的,该机制由融合剂 gB 和受体结合复合物 gH/gL 组成。异二聚体 gH/gL 复合物与 EBV 上皮细胞受体或 gp42 结合,后者与 B 细胞受体结合,触发 gB 介导的病毒体包膜与细胞膜的融合。我们之前的研究发现gL糖基化突变体N69L/S71V具有上皮细胞特异性的超融合表型。为了研究这种 gL 突变体对 gB 驱动的上皮细胞融合的启动和动力学的影响,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,可以使用活细胞实时测量膜融合。从早期时间点开始,gL_N69L/S71V 突变体的上皮细胞融合活性大幅增加,比野生型 gL 高出 300%。gL 突变体的超融合性并不是 gH 复合物形成改变或细胞定位改变的结果。此外,gL 突变体的超融合表型与增大的合胞体的形成相关。总之,我们目前的研究结果强调了 gL 在 gB 介导的上皮细胞融合动力学中的重要作用,补充了先前的发现,表明 EBV 膜融合中 gL 和 gB 之间存在直接相互作用。

重要性EBV 主要感染上皮细胞和 B 淋巴细胞,它们是 EBV 相关恶性肿瘤霍奇金淋巴瘤、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发挥着最难以捉摸的作用。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,我们的数据表明,gL 糖基化突变体增加了超融合 gB 突变体的融合活性,表明该 gL 突变体和 gB 突变体在融合过程中针对不同的步骤。我们对 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 EBV 对上皮细胞和 B 淋巴细胞的高度调节趋向性至关重要,并可能产生防止病毒进入靶宿主细胞的新治疗策略。最后,使我们的结果特别令人感兴趣的是其他疱疹病毒中不存在 gL 合胞体突变体。

更新日期:2017-11-15
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