Mutagenesis of the signal sequence of yellow fever virus prM protein: Enhancement of signalase cleavage in vitro is lethal for virus production
Mutagenesis of the signal sequence of yellow fever virus prM protein: Enhancement of signalase cleavage in vitro is lethal for virus production
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DOI：
10.1128/jvi.74.1.24-32.2000
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发表时间：
2000-01-01
影响因子：
5.4
通讯作者：
Lobigs, M
中科院分区：
文献类型：
--
作者：
Lee, E;Stocks, CE;Lobigs, M
黄病毒多蛋白中C-prM连接处的蛋白水解加工涉及内部信号序列的胞质和腔侧的协调切割。我们在黄热病病毒（YFV）prM信号序列的COOH末端引入了氨基酸取代（VPQAQA突变），其将prM蛋白的有效信号肽酶切割与其对病毒NS 2B-3蛋白酶介导的C蛋白在细胞质中的先前切割的依赖性解偶联，用全长YFV RNA转录物的亲和性测定显示VPQAQA突变，其在体外增强信号肽酶切割，对于感染性病毒生产是致命的。从用VPQAQA RNA转染的细胞中回收回复突变体或第二位点突变体。这些病毒的分析表明，在不同的结构域的prM信号序列中的单个氨基酸取代可以恢复活力，这些变体在脊椎动物细胞中的生长特性，仅略有不同的亲本病毒，尽管有效的信号肽酶切割prM在无细胞表达试验。然而，VPQAQA变体在小鼠中的神经毒力显著减弱。这项研究表明，在prM信号序列中的取代，破坏协调裂解在C-prM交界处可以冲击突变病毒的生物学特性。除了prM的生产速率以外的因素都是由该位点上的调节性裂解来控制的。
Protcolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease, Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production, Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability, These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.