Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH
Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH
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DOI：
10.1128/jvi.00084-18
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发表时间：
2018-02
影响因子：
5.4
通讯作者：
M. Vallbracht;Sascha Rehwaldt;B. Klupp;T. Mettenleiter;W. Fuchs
中科院分区：
文献类型：
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作者：
M. Vallbracht;Sascha Rehwaldt;B. Klupp;T. Mettenleiter;W. Fuchs
摘要许多病毒包膜蛋白被天冬酰胺(N)连接的糖基化修饰，这会影响其结构、理化性质、细胞内转运和功能。在这里，我们系统地分析了甲型疱疹病毒伪狂犬病病毒(PRV)糖蛋白H(Gh)中N-连接的糖链的功能相关性，Gh是保守的核心疱疹病毒融合机制的重要组成部分。在Gd介导的受体结合后，Gh和Gl的异二聚体复合体激活Gb，介导病毒被膜与宿主细胞膜的融合，以便病毒进入。GH在77、162、542、604和627位含有5个潜在的N连接糖基化位点，它们可以被保守突变(天冬酰胺到谷氨酰胺)单独或组合失活。对突变蛋白进行正确表达和融合活性检测。此外，突变的Gh基因被插入到PRV基因组中，用于分析病毒感染过程中的功能。我们的结果表明，所有五个位点都是糖基化的。灭活PRV特异性N77或保守的N627导致体外融合活性显著降低，穿透动力学延迟，病毒斑变小。此外，N627的替换极大地影响了Gh在转基因细胞中的转运，导致内质网(ER)滞留和表面表达减少。相比之下，在水痘病毒属中保守的N604突变导致了体外融合活性和病毒细胞间传播的增强。这些结果表明N-糖链在PRV-Gh的正确定位和功能中起作用。然而，即使同时灭活Gh的所有五个N-糖基化位点，也不能严重抑制感染性病毒颗粒的形成。重要意义疱疹病毒感染需要病毒被膜与细胞膜的融合，这涉及到由gB和异源二聚体Gh/gl复合体组成的保守的融合机制。真正的融合蛋白Gb依赖于Gh/Gl复合体的存在而激活。病毒包膜糖蛋白，如Gh，通常含有N-糖链，这对它们的折叠、运输和功能有很大的影响。在这里，我们系统地分析了所有五个预测的甲型疱疹病毒伪狂犬病病毒(PRV)Gh中N-连接糖基化位点的功能相关性。尽管特定位点的突变影响了Gh的转运、体外融合活性和细胞到细胞的扩散，并导致了延迟的穿透动力学，但即使Gh的所有5个N-糖基化位点同时失活，也不能严重抑制感染性病毒颗粒的形成。因此，我们的结果证明了N-聚糖对Gh功能的调节作用，但不是必需的。
ABSTRACT Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport, in vitro fusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.