猪繁殖与呼吸综合征病毒核衣壳蛋白翻译起始位点的研究

Translational Control of Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus

猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)核衣壳蛋白(Nucleocapsid protein,N蛋白)的表达是通过非连续性转录合成的亚基因组(Subgenomic,sg)mRNA翻译而来。但位于亚基因组前导序列中的2个AUG均不能作为N蛋白翻译的起始位点,其翻译使用的是N开放阅读框(ORF)中的首个AUG,因此,本研究旨在感染性克隆的基础上研究N蛋白的翻译起始位点。作者实验室已有在ORF6和ORF7之间插入酶切位点的感染性克隆pORF673,该突变克隆中含有3个潜在的AUG,分别构建了pORF673中N蛋白的其中2个潜在的起始密码子AUG突变的全长克隆,转染Marc-145细胞。这些突变体都能够拯救出子代病毒,其中2个突变病毒的N蛋白的前11个氨基酸完全改变,这些突变病毒都能够在Marc-145细胞上稳定传代。经RT-PCR分析子代病毒的N基因序列及其亚基因组序列,结果表明N蛋白的翻译偏向于使用其ORF的第1个潜在的AUG。如果后者移码,则病毒可以自身修复,这是首次发现病毒有矫正ORF的功能。本研究为N蛋白N端插入标签作为标记疫苗以及进一步研究N蛋白结构与功能奠定了基础。

The structural genes of PRRSV are expressed from a nested set of subgenomic mRNAs（sgmRNA）.Nucleocapsid（N） protein is expressed from sgmRNA7,but the initiation site of translation is the first AUG in N coding region,not the AUG in leader sequence of sgmRNA.This experiment is designed to study the translational initiation site of N protein.pORF673 is a former constructed full-length infectious cDNA clone that separated ORF6 ORF7 and inserted three restriction sites,the conjunction sequence contains three potential AUGs.Three full-length cDNA clones encompassing ATG mutation were conducted to estimate their effects on virus replication.Viable viruses could be recovered and stably passaged on Marc-145 cells.The first 11 residues of two recovered viruses were fully changed.The result indicates that the expression of N protein prones to choose the first AUG.This study lays a foundation for the genetically tagged vaccine and further molecular dissection of the roles of N protein in PRRSV replication cycle.