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.

中文翻译：

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病毒亚基因组mRNA合成需要2型PRRSV的Nsp12编码区。

作为猪繁殖与呼吸综合征病毒（PRRSV）的许多非结构蛋白之一，非结构蛋白12（Nsp12）受到的关注相对较少，其在病毒复制中的作用（如果有的话）基本上是未知的。通过对传染性PRRSV克隆进行逆向遗传操作，当前的研究首次证明Nsp12是PRRSV复制的关键组成部分。此外，对Nsp12的生化特性进行了评估，发现Nsp12暴露于氧化条件下会形成二聚体。此外，我们系统地分析了Nsp12在PRRSV RNA合成中使用链特异性PCR方法的功能。令我们惊讶的是，未发现Nsp12参与负链基因组RNA（-gRNA）的合成。重要的，我们的结果表明Nsp12参与正链和负链亚基因组mRNA（+ sgmRNA和-sgmRNA）的合成。最后，我们发现sgmRNA合成需要Nsp12中的半胱氨酸35和半胱氨酸79的组合。据我们所知，我们是第一个报道Nsp12在PRRSV生命周期中的生物学作用的人，我们得出的结论是Nsp12与+ sgRNA和-sgRNA的合成有关。