猪繁殖和呼吸综合征病毒GP3蛋白糖基化位点突变株的构建及其生物学特性分析

FUNCTIONAL ANALYSIS OF GLYCOSYLATION OF GP3 PROTEIN OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

为研究猪繁殖与呼吸系统综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)GP3蛋白糖基化位点的作用,在弱毒疫苗株HuN4-F112感染性克隆的基础上,采用点突变的方法对GP3蛋白N29、N42、N50、N131位进行单点或多点组合突变去糖基化,构建了不同糖基化位点的单点和多点突变的PRRSV全长cDNA克隆,最终成功救获N29Q、N42Q、N50Q、N131Q、N29Q/N50Q、N29Q/N195Q六株含糖基化位点突变的病毒,而其余几种糖基化位点突变组合的全长cDNA未能拯救出病毒。对拯救毒株进行滴度测定及多步生长曲线绘制等特性分析,结果显示N29Q、N42Q、N50Q、N131Q位单个糖基化位点的缺失虽不影响子代病毒的产生但会不同程度地降低PRRSV感染细胞的能力,其中N50Q突变体较亲本下降6个滴度且生长明显延迟。多个糖基化位点同时缺失则会影响病毒的拯救,推测这些糖基化位点可能是产生具有感染性病毒粒子所不可或缺的。

To analyze the function of glycosylation of GP3 protein in Porcine reproductive and respiratory syndrome virus（PRRSV）,site-directed mutagenesis was used to introduce individual amino acid substitutions into an infectious cDNA clone of the attenuated vaccine strain HuN4-F112.A series of cDNA clones with mutations of N→Q at glycosylation sites of GP3 protein were constructed.Six clones with single mutation as N29Q,N42Q,N50Q,N131Q,or double mutations as N29Q/N50Q and N29Q/N195Q were successfully rescued,while other clones with multiple mutations were all failed to rescue viruses.The results demonstrated that deglycosylation of sites N29,N42,N50,N131 had no influence on progeny virus production but could affect virus titers in variant degree,especially the N50Q,whose titer was 106-fold lower than its parental strain,and showed an obvious growth delay.The outcome of this study may lay a basis for further study on the relationship between glycosylation and virulence of PRRSV.