ATTENUATED MUTANTS OF VENEZUELAN EQUINE ENCEPHALITIS-VIRUS CONTAINING LETHAL MUTATIONS IN THE PE2 CLEAVAGE SIGNAL COMBINED WITH A 2ND-SITE SUPPRESSOR MUTATION IN E1
ATTENUATED MUTANTS OF VENEZUELAN EQUINE ENCEPHALITIS-VIRUS CONTAINING LETHAL MUTATIONS IN THE PE2 CLEAVAGE SIGNAL COMBINED WITH A 2ND-SITE SUPPRESSOR MUTATION IN E1
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DOI：
10.1006/viro.1995.1458
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发表时间：
1995-09-10
期刊：
影响因子：
3.7
通讯作者：
JOHNSTON, RE
中科院分区：
文献类型：
--
作者：
DAVIS, NL;BROWN, KW;JOHNSTON, RE
通过定点诱变消除甲病毒委内瑞拉马脑炎病毒（VEE）的全长cDNA克隆中的PE 2切割信号。来自所得质粒的RNA转录物在转染幼仓鼠肾（BHK）细胞后编程产生非存活颗粒。然而，突变RNA也产生了小比例的活回复突变体。对这些生物回复突变体及其分子克隆同源物的分析表明，E2位置243或E1位置253处的第二位点抑制突变能够恢复PE 2切割信号突变体的活力。活的回复突变体将未处理的PE 2掺入颗粒中，其显示出对BHK细胞的正常感染性，但降低了在C6/36蚊子细胞中生长的能力。携带致死性PE 2切割信号突变的突变体与El 253处的抑制子组合，当通过皮下、脑内或鼻内途径接种时，在成年小鼠中是无毒力的或高度减毒的，并且赋予针对用毒性VEE的腹膜内和鼻内攻击的完全保护。这些结果表明甲病毒刺突中E2和E1蛋白的密切功能关联。它们还对VEE、其他甲病毒和使用类似糖蛋白成熟机制的其他病毒的重组活病毒疫苗的设计具有意义。(C)出版社：Academic Press
The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a smalt proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at El 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus Vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation. (C) 1995 Academic Press, Inc.