The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

作为猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus, PRRSV）的众多非结构蛋白之一，非结构蛋白12（nonstructural protein 12, Nsp12）迄今受到的关注相对较少，其在病毒复制中所发挥的作用（若确实存在）基本尚未明确。本研究借助感染性PRRSV克隆的反向遗传操作技术，首次证实Nsp12是PRRSV复制过程中的关键组成部分。此外，本研究对Nsp12的生化特性进行了评估，结果显示Nsp12在氧化环境下可形成二聚体。进一步地，本研究采用链特异性PCR（strand-specific PCR）技术，系统性分析了Nsp12在PRRSV RNA合成过程中的功能。令人意外的是，本研究未发现Nsp12参与负链基因组RNA（minus-strand genomic RNA, -gRNA）的合成；值得注意的是，实验结果表明Nsp12同时参与正链与负链亚基因组mRNA（plus- and minus-strand subgenomic mRNAs, +sgmRNA与-sgmRNA）的合成。最后，本研究发现，Nsp12中第35位与第79位半胱氨酸的协同作用是亚基因组mRNA（sgmRNA）合成所必需的。据我们所知，本研究是首次报道Nsp12在PRRSV生命周期中的生物学功能，并最终得出结论：Nsp12同时参与正链与负链亚基因组RNA（+sgRNA与-sgRNA）的合成。