Genzentrum, Max-von-Pettenkofer-Institut, Lehrstuhl Virologie, Ludwig-Maximilians-Universität (LMU), München, Germany.
PLoS One. 2007 May 23;2(5):e459. doi: 10.1371/journal.pone.0000459.
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.
严重急性呼吸综合征冠状病毒 (SARS-CoV) 的基因组预计编码 14 个功能开放阅读框，导致多达 30 种结构和非结构蛋白产物的表达。大量病毒 ORF 的功能了解甚少或未知。为了更深入地了解不同蛋白质的功能、作用方式和相互作用，我们克隆了病毒 ORFeome 并进行了全基因组分析，以研究病毒内蛋白相互作用和细胞内定位。通过酵母双杂交矩阵分析测试了 900 对成对相互作用，鉴定出 65 个以上的阳性非冗余相互作用，包括 6 个自相互作用。随后，约 38%的相互作用通过哺乳动物细胞中的 CoIP 得到了验证。Nsp2、nsp8 和 ORF9b 与其他病毒蛋白表现出广泛的相互作用。Nsp8 与复制酶蛋白 nsp2、nsp5、nsp6、nsp7、nsp8、nsp9、nsp12、nsp13 和 nsp14 相互作用，表明其作为复制复合物机制中的主要参与者发挥着关键作用。其他人已经表明，nsp8 在体外病毒复制中是必需的，而 nsp2 不是。我们表明，辅助蛋白 ORF9b 对于病毒复制也没有关键作用，因为它可以从病毒中缺失，而病毒在 Vero 细胞中显示出正常的蚀斑大小和生长特征。然而，由于其大量的相互作用，它可能对病毒-宿主相互作用和致病性很重要，通过增强 SARS 蛋白组网络的整体稳定性，或者在调节蛋白-蛋白相互作用方面发挥一些未实现的作用。鉴定出的相互作用为未来的研究提供了有价值的材料。
