Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus
Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus
复制标题
绘制猪繁殖与呼吸综合征病毒的非结构蛋白相互作用网络。
DOI：
10.1128/jvi.01112-18
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发表时间：
2018-12-01
影响因子：
5.4
通讯作者：
Yang, Hanchun
中科院分区：
文献类型：
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作者：
Song, Jiangwei;Liu, Yuanyuan;Yang, Hanchun
猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）是一种正链RNA病毒，属于动脉炎病毒科。病毒RNA的合成由复制/转录复合物（RTC）指导，所述复制/转录复合物主要由PRRSV非结构蛋白（nsps）和可能的细胞蛋白的网络组成。在这里，我们映射的相互作用网络之间的PRRSV nsps通过使用酵母双杂交筛选结合免疫共沉淀（co-IP）和共转染试验。我们总共鉴定了24种新的相互作用，并发现这些相互作用集中在开放阅读框架1b（ORF 1b）编码的nsps上，这些nsps主要由跨膜蛋白nsp 2、nsp 3和nsp 5连接。有趣的是，核心酶nsp 9和nsp 10与跨膜蛋白的相互作用并不是以直接的方式发生的，因为它们在co-IP测定中起作用，但在完整的哺乳动物细胞中发现彼此的能力很差。进一步证明它们可以在细胞内相互作用，需要对nsp 9和nsp 10的N端截短进行工程改造。然而，尽管共转染细胞中的共定位关系较差，但nsp 9和nsp 10都与膜蛋白（例如，nsp 2）在PRRSV感染的细胞中的病毒复制和转录复合物（RTC）处。因此，我们的研究结果表明，存在一个复杂的相互作用网络之间的PRRSV nsps和提高的可能性，招聘的关键复制酶蛋白膜相关的nsps可能涉及一些监管机制在infection.IMPORTANCE宿主细胞内的PRRSV RNA的合成取决于RTC的有效和正确的组装，发生在修饰的细胞内膜。作为解剖这一知之甚少的事件的重要一步，我们调查了PRRSV nsps之间的相互作用网络。我们的研究建立了一个全面的互动地图的PRRSV nsps，并揭示了网络中的重要球员。结果还强调了在PRRSV RTC组装期间，可能存在PRRSV核心酶nsp 9和nsp 10向病毒膜nsps的受调节的募集。
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the family Arteriviridae. Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.IMPORTANCE Synthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.