Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus
Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus
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DOI：
10.1128/jvi.00690-12
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发表时间：
2012-08-01
影响因子：
5.4
通讯作者：
Mettenleiter, Thomas C.
中科院分区：
文献类型：
--
作者：
Fuchs, Walter;Backovic, Marija;Mettenleiter, Thomas C.
糖蛋白 H (gH) 是疱疹病毒科中保守的包膜蛋白。 gH 和糖蛋白 L (gL) 的异二聚体复合物与糖蛋白 B (gB) 一起介导渗透并指导病毒在细胞间传播。在单纯疱疹病毒和伪狂犬病病毒 (PrV) 中，gH/gL、gB 和 gD 的共表达诱导膜融合形成多核细胞。最近确定的 PrV gH 核心片段的晶体结构揭示了与其他 gH 蛋白的显着结构相似性（M. Backovic 等人，Proc. Natl. Acad. Sci. U. S. A. 107:22635-22640,2010）。在膜近端部分（结构域 IV）内，保守的带负电的表面环（瓣）两侧是分子内二硫键。该瓣与 N 连接的碳水化合物部分一起覆盖了下面的疏水残基块。为了研究这些结构的功能相关性，通过定点诱变引入非保守氨基酸取代。测试突变蛋白的正确表达、融合活性以及 gH 缺失的 PrV 的功能互补。皮瓣和疏水片内的几个单一氨基酸变化是可以耐受的，糖基化位点的删除仅产生很小的影响。然而，皮瓣或疏水片内的多个丙氨酸取代导致了显着的缺陷。 gH 功能也受到皮瓣 C 末端二硫键破坏以及引入旨在桥接皮瓣中心部分与疏水补片的半胱氨酸对后的严重影响。有趣的是，所有突变的 gH 蛋白都能补充 gH 缺失的 PrV，但融合缺陷的 gH 突变体导致病毒进入明显延迟。
Glycoprotein H (gH) is an envelope protein conserved in the Herpesviridae. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635-22640,2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.