Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site
Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site
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DOI：
10.1128/jvi.01499-10
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发表时间：
2010-12-01
影响因子：
5.4
通讯作者：
Tong, Shuping
中科院分区：
文献类型：
--
作者：
Ito, Kiyoaki;Qin, Yanli;Tong, Shuping
乙肝病毒包膜基因S区域的突变与免疫逃逸、隐匿性感染和治疗抵抗有关。我们之前发现了S基因自然发生的突变，这种突变会改变乙肝病毒的病毒粒子分泌。在这里，我们用反互补实验证实了病毒被膜蛋白S结构域的I110M、G119E和R169P突变抑制了病毒粒子的分泌，而M133T突变挽救了I110M和G119E突变株的病毒粒子分泌。G119E突变损害了分泌型乙肝表面抗原(HBs)的检测，提示免疫逃逸。突变蛋白R169P也不能分泌乙肝表面抗原，与野生型包膜蛋白共表达时具有显性负效应。虽然S结构域存在于所有三种包膜蛋白中，但I110M、G119E和R169P突变会损害病毒通过小包膜蛋白分泌病毒粒子。相反，仅共表达M133T突变体的小包膜蛋白就可以挽救病毒粒子的分泌。M133T突变还可以克服G145R免疫逃逸突变或N-连接糖基化位点N146突变引起的分泌缺陷。事实上，M133T突变创建了一个新的N-连接糖基化位点((NST133)-N-131)。用N131Q/T突变破坏该位点或用衣霉素处理细胞来阻止糖基化均可消除M133T突变的作用。我们的发现表明，乙肝病毒包膜蛋白的N-连接糖基化对病毒粒子的分泌是至关重要的，而由S蛋白突变引起的分泌缺陷可以通过额外的糖基化位点来挽救。
Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((NST133)-N-131). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.