Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion
Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion
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DOI：
10.1128/jvi.00376-18
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发表时间：
2018-04
影响因子：
5.4
通讯作者：
M. Vallbracht;W. Fuchs;B. Klupp;T. Mettenleiter
中科院分区：
文献类型：
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作者：
M. Vallbracht;W. Fuchs;B. Klupp;T. Mettenleiter
疱疹病毒膜融合依赖于由糖蛋白B （gB）和gH/gL组成的核心融合机制。虽然gB在结构上类似于自主的III类融合蛋白，但它严格依赖gH/gL来驱动膜融合。gH/gL复合物是否需要通过膜锚定来实现其功能，以及gH细胞质（CD）和跨膜结构域（TMD）在融合中发挥何种作用尚不清楚。虽然gH CD和TMD在感染过程中发挥重要作用，但在短暂的试验中，单纯疱疹病毒1 （HSV-1）的可溶性gH/gL似乎足以介导细胞-细胞融合，这与CD和TMD的重要贡献相矛盾。为了进一步阐明这种明显的差异，我们研究了相关的甲型疱疹病毒伪狂犬病毒gH的CD和TMD的作用。为此，我们表达了c端截断的可溶性gH，并用糖基磷脂酰肌醇（gpi）锚点代替TMD。我们还生成了含有PrV gD或HSV-1 gH的TMD和/或CD的嵌合体。在基于细胞的融合实验和病毒感染过程中对蛋白质进行了表征。虽然CD的截断导致膜融合活性降低，但突变蛋白仍然支持gH阴性PrV的复制，这表明PrV gH CD对于病毒复制是不可或缺的。相比之下，缺乏TMD的PrV gH，通过脂质连接物膜锚定的PrV gD TMD，或包含PrV gD TMD的PrV gH是非功能性的，突出了gH TMD在功能中的重要作用。有趣的是，尽管序列一致性较低，HSV-1 gH TMD可以替代PrV gH TMD，这表明功能守恒。包膜病毒依靠膜融合进入。虽然这一过程只能由一种或两种蛋白质介导，但疱疹病毒依赖于至少三种不同糖蛋白的协同作用。虽然gB具有真正融合蛋白的特征，但它依赖于gH及其复合伙伴gL进行融合。gH/gL是阻止过早融合还是主动触发gb介导的融合尚不清楚，gH/gL的功能是否需要稳定的膜锚定，还是单靠外畴就足够了，目前的结果也存在矛盾。我们的研究结果表明，在假狂犬病毒gH中，跨膜锚点在gb介导的融合中起着至关重要的作用，而细胞质尾巴则不是严格要求的。
ABSTRACT Herpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation. IMPORTANCE Enveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.