Lv Huifang, Dong Wang, Qian Gui, Wang Jie, Li Xiaomeng, Cao Zhi, Lv Qizhuang, Wang Chengbao, Guo Kangkang, Zhang Yanming
College of Veterinary Medicine, Northwest A&F University, No. 22 Xinong Road, Yangling 712100, Shaanxi, PR China.
Qingdao Yebio Biological Engineering Co. Ltd. (Yebio), No. 21 Aodongnan Road, Qingdao 266114, Shandong, PR China.
J Gen Virol. 2017 Jul;98(7):1679-1692. doi: 10.1099/jgv.0.000867. Epub 2017 Jul 19.
Classical swine fever (CSF) is a severe, febrile and highly contagious disease caused by classical swine fever virus (CSFV) that has resulted in huge economic losses in the pig industry worldwide. CSFV Npro has been actively studied but remains incompletely understood. Few studies have investigated the cellular proteins that interact with Npro and their participation in viral replication. Here, the yeast two-hybrid (Y2H) system was employed to screen Npro-interacting proteins from a porcine alveolar macrophage (PAM) cDNA library, and a blast search of the NCBI database revealed that 15 cellular proteins interact with Npro. The interaction of Npro with ribosomal protein S20, also known as universal S10 (uS10), was further confirmed by co-immunoprecipitation and glutathione S-transferase pull-down assays. Furthermore, uS10 overexpression inhibited CSFV replication, whereas the knockdown of uS10 promoted CSFV replication in PAMs. In addition, Npro or CSFV reduced uS10 expression in PAMs in a proteasome-dependent manner, indicating that Npro-uS10 interaction might contribute to persistent CSFV replication. Our previous research showed that CSFV decreases Toll-like receptor 3 (TLR3) expression. The results showed that uS10 knockdown reduced TLR3 expression, and that uS10 overexpression increased TLR3 expression. Notably, uS10 knockdown did not promote CSFV replication following TLR3 overexpression. Conversely, uS10 overexpression did not inhibit CSFV replication following TLR3 knockdown. These results revealed that uS10 inhibits CSFV replication by modulating TLR3 expression. This work addresses a novel aspect of the regulation of the innate antiviral immune response during CSFV infection.
经典猪瘟（CSF）是一种由经典猪瘟病毒（CSFV）引起的严重、发热且高度传染性的疾病，已在全球养猪业中造成巨大经济损失。对CSFV Npro已进行了积极研究，但仍未完全了解。很少有研究调查与Npro相互作用的细胞蛋白及其在病毒复制中的作用。在此，利用酵母双杂交（Y2H）系统从猪肺泡巨噬细胞（PAM）cDNA文库中筛选与Npro相互作用的蛋白，对NCBI数据库进行的blast搜索显示有15种细胞蛋白与Npro相互作用。通过免疫共沉淀和谷胱甘肽S-转移酶下拉试验进一步证实了Npro与核糖体蛋白S20（也称为通用S10，即uS10）之间的相互作用。此外，uS10的过表达抑制了CSFV复制，而在PAM中敲低uS10则促进了CSFV复制。此外，Npro或CSFV以蛋白酶体依赖性方式降低了PAM中uS10的表达，表明Npro-uS10相互作用可能有助于CSFV的持续复制。我们之前的研究表明CSFV会降低Toll样受体3（TLR3）的表达。结果显示，敲低uS10会降低TLR3的表达，而过表达uS10则会增加TLR3的表达。值得注意的是，在过表达TLR3后，敲低uS10并不会促进CSFV复制。相反，在敲低TLR3后，过表达uS10也不会抑制CSFV复制。这些结果表明，uS10通过调节TLR3的表达来抑制CSFV复制。这项工作揭示了CSFV感染期间先天抗病毒免疫反应调节的一个新方面。
