PCV2 Rep基因启动子区vISRE突变株的生物学特性

Biological character of porcine circovirus type 2 with site mutation at interferon stimulation reaction element(ISRE) like region in Rep gene promoter

【目的】为了初步揭示PCV2Rep基因启动子区类干扰素刺激反应元件(vISRE)的生物学功能。【方法】应用感染性克隆技术构建了2株vISRE点突变的重组PCV2,对突变病毒在PK15细胞上的增殖特性、遗传稳定性及对干扰素刺激的反应特性进行了分析。【结果】Rep基因启动子区vISRE点突变后PCV2仍可在PK15细胞中正常复制,但病毒滴度比亲本毒株下降。PCV21740G-C在PK15细胞上3至10代之间遗传稳定,PCV21741A-T在PK细胞上第3代病毒保持突变基因的特征,但传至第7代时1743和1744位的AC突变为TT,并一直保持到第10代。100U/mL的PoIFN-α处理感染病毒的PK15细胞后,亲本毒株和2个突变毒株的阳性感染细胞数量均有增加,但亲本毒株病毒粒子数的增加显著高于2个突变毒株。【结论】Rep基因启动子区vISRE的突变影响PCV2在PK15上的增殖和对干扰素刺激的反应,推测其可能在干扰素促进病毒增殖中发挥调控作用。

[ Objective ] To explore the biological function of the interferon stimulation reaction element （ISRE） like motif CTGAAAACGAAAGA within porcine circovirus type 2 （PCV2） Rep promoter. [ Methods I Two recombinant PCV2 strains, namely PCV2 1740^G-C and PCV2 1741^A-T, were constructed by transfecting PK15 cells with site-mutated infectious clone of PCV2 strain Denta. Replication character, genetic stability and reactive character to porcine interferon alpha （poIFN-α） were compared among parental PCV2 and the two mutant viruses. [ Results ] The ISRE like motif in Rep promoter was not necessary for the replication of PCV2 because two site-mutated viral genome clones both produced infectious virus. In contrast to parental PCV2, the viral antigen positive PK15 ceils of the two site-mutated PCV2 were decreased. PCV2 1740^G-c was genetically stable in the PK15 cell while PCV2 1741AT was found to have another two nucleotide mutated from 1744AC1745 to 1744TY1745 between 3^th and 7^th passage in the PK15 cell. After treated with 100 U/mL porcine interferon alpha, the viral antigen positive PK15 cells and virus genomes of parental PCV2 and two site-mutated viruses were both increased. But the enhancement rate of the two sitemutated PCV2 was significantly lower than parental PCV2. [ Conclusion] Site-mutation of ISRE like motif in Rep promoter decreased the replication and poIFN-α induced enhancement of PCV2 in PK15 cells. According to these above results, it maybe speculated that ISRE like motif in PCV2 Rep gene promoter contain a functional element and it may contribute to the interferon inducible enhancement of virus replication in PK15 ceils.