Department of Animal Science, North Carolina State University, Raleigh, NC, USA.
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Virus Res. 2018 Apr 2;249:85-92. doi: 10.1016/j.virusres.2018.03.010. Epub 2018 Mar 22.
Porcine reproductive and respiratory syndrome (PRRS) is characterized by abortions in pregnant sows and respiratory disease, particularly in young pigs. The causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family. GP5 and M are the major envelope proteins encoded by PRRSV. To further characterize these two viral proteins, a yeast two-hybrid approach was utilized to identify interacting partners of PRRSV GP5 and M proteins.
Interacting partners of PRRSV GP5 and M were identified using a porcine macrophage cDNA library yeast two-hybrid screen. Subsequently, the interactions between PRRSV GP5/M and the cellular protein Snapin were mapped using truncated versions of the GP5 and M proteins in a yeast two-hybrid assay to localize the interactions. The Snapin gene from the African green monkey kidney cell line MARC-145, which is permissive to PRRSV, was cloned and sequenced, and compared to porcine Snapin. Cellular Snapin expression was reduced in PRRSV-infected cells via Snapin-specific siRNA targeting.
Here we show that the cellular Snap-Associated Protein (Snapin), an accessory protein of the SNARE membrane fusion network and also a member of the BLOC-1 complex, specifically interacts with GP5 and M. Inhibition of Snapin expression via siRNA targeting of Snapin results in the reduction of PRRSV replication.
The PRRSV GP5 and M proteins are known to form a heterodimeric complex which is important for viral structure and infectivity, and both PRRSV proteins can interact with cellular Snapin. Snapin knock-down suggests these interactions could be important in the PRRSV lifecycle. GP5 and M proteins may interact with Snapin to exploit its roles in intracellular transport and membrane fusion.
猪繁殖与呼吸综合征（PRRS）的特征是妊娠母猪流产和呼吸道疾病，尤其是在小猪中。病原体是猪繁殖与呼吸综合征病毒（PRRSV），是动脉病毒科的一员。GP5 和 M 是 PRRSV 编码的主要包膜蛋白。为了进一步表征这两种病毒蛋白，使用酵母双杂交方法鉴定了 PRRSV GP5 和 M 蛋白的相互作用伙伴。
使用猪巨噬细胞 cDNA 文库酵母双杂交筛选鉴定 PRRSV GP5 和 M 的相互作用伙伴。随后，使用 GP5 和 M 蛋白的截短版本在酵母双杂交测定中绘制 PRRSV GP5/M 与细胞蛋白 Snapin 之间的相互作用，以定位相互作用。从允许 PRRSV 感染的非洲绿猴肾细胞系 MARC-145 克隆并测序 Snapin 基因，并与猪 Snapin 进行比较。通过针对 Snapin 的特异性 siRNA 靶向降低 PRRSV 感染细胞中的细胞 Snapin 表达。
我们证明细胞 Snap 相关蛋白（Snapin），即 SNARE 膜融合网络的辅助蛋白，也是 BLOC-1 复合物的成员，与 GP5 和 M 特异性相互作用。通过针对 Snapin 的 siRNA 靶向抑制 Snapin 表达会降低 PRRSV 复制。
PRRSV GP5 和 M 蛋白已知形成异二聚体复合物，这对于病毒结构和感染力很重要，并且两种 PRRSV 蛋白都可以与细胞 Snapin 相互作用。Snapin 敲低表明这些相互作用在 PRRSV 生命周期中可能很重要。GP5 和 M 蛋白可能与 Snapin 相互作用以利用其在细胞内运输和膜融合中的作用。
