Tan Terence T T, Bhuvanakantham Raghavan, Li Jun, Howe Josephine, Ng Mah-Lee
Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, 5 Science Drive 2, National University of Singapore, Singapore 117597.
J Gen Virol. 2009 May;90(Pt 5):1081-1092. doi: 10.1099/vir.0.007872-0. Epub 2009 Mar 4.
Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative trans complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation.
黄病毒前体膜（prM）蛋白在包膜（E）蛋白的构象折叠中起重要作用，并保护其免受高尔基体网络酸性囊泡中过早融合的影响。目前，prM蛋白胞外域上介导黄病毒组装过程中关键步骤的分子决定因素尚不明确。在本研究中，生物信息学分析和丙氨酸扫描诱变表明，氨基酸三联体缬氨酸76、酪氨酸78和甘氨酸79在黄病毒prM胞外域中绝对保守。在西尼罗河病毒（WNV）的prM胞外域中这些残基处设计的三重突变完全消除了病毒感染性。prM蛋白的定点诱变表明，氨基酸三联体中的酪氨酸78是病毒感染性和分泌所必需的。该突变不影响prM和E蛋白的折叠、翻译后修饰及运输。使用透射电子显微镜进行的超微结构研究证实，酪氨酸78突变阻断了病毒颗粒的形成。阳性和阴性反式互补研究证明了酪氨酸78突变赋予的组装缺陷的特异性。总体而言，这些结果将酪氨酸78定义为prM蛋白胞外域上存在的一种新型关键决定因素，是黄病毒组装所必需的。prM蛋白功能的分子剖析为阐明黄病毒颗粒形成提供了急需的关键知识。
