猪伪狂犬病毒EP0蛋白拮抗宿主蛋白Spindlin1抗病毒作用的研究

PRV EP0 protein antagonizes the antiviral activity of host protein Spindlin1

为了分析Spindlin1 (Spin1)蛋白对猪伪狂犬病毒(PRV)复制的影响,本研究利用GE Dharmacon siRNA网站设计3条Spin1 si RNA (siSpin1-376、siSpin1-537与si Spin1-654),将其以1∶1∶1的比例混合,以30 pmol的剂量转染PK-15细胞,转染后48 h感染PRV He N1株,并分别于感染后6 h、12 h、18 h和24 h收集细胞及上清样品,进行病毒的TCID50测定,结果显示,与对照组相比,感染后12 h,干扰Spin1的表达能够显著促进PRV在PK-15细胞中的增殖(P<0.05)。将PRV以MOI 0.1感染PK-15细胞后不同时间,利用western blot检测PRV感染对Spin1蛋白表达水平的影响,结果显示,与转染siRNA-NO的对照组相比,PRV感染后12 h细胞中的Spin1蛋白表达水平显著下降(P<0.05),其余时间段与对照组均无显著差异。为了阐明PRV拮抗Spin1抗病毒作用的分子机制,本研究分别构建PRV早期蛋白EP0与Spin1的真核表达质粒并分别经PCR及测序鉴定正确后共转染人胚胎肾细胞(HEK293FT),western blot结果显示,与单独转染Spin1表达质粒的细胞相比,共转染Spin1与EP0表达质粒细胞中Spin1的表达水平明显下调。进一步利用免疫共沉淀试验(Co-IP)分析二者的相互作用情况,结果显示,EP0与Spin1在共转染的细胞中存在相互作用。上述结果表明,PRV EP0蛋白能够通过与Spin1蛋白的相互作用下调其的表达。本研究发现了一个新的能够抑制PRV复制的宿主蛋白,并阐明了PRV拮抗宿主抗病毒因子的一种新机制,为揭示PRV的致病机理以及新型药物的研发提供参考。

In order to study the effect of Spindlin1(Spin1) protein on pseudorabies virus(PRV) replication,three Spin1si RNAs(siSpin1-376,si Spin1-537 and si Spin1-654) were designed using GE Dharmacon si RNA design website in this study.The si RNAs were mixed at 1 ∶1 ∶1 ratio and transfected into PK-15 cells at a dose of 30 pmol,and then the cells were infected with PRV-HeN1 strain at 48 hours after transfection,and the cell and supernatant samples were collected at 6,12,18 and 24 hpi for TCID50analysis.The results showed that the interference of Spin1 could significantly promote the proliferation of PRV at 12 hpi.PK-15 cells were infected with PRV,and then the effect of PRV infection on Spin1 expression was analyzed by western blot.The results showed that the expression level of Spin1 protein in PRV-infected cells was significantly decreased compared with the control group.To elucidate the molecular mechanism of PRV antagonizing the antiviral activity of Spin1,the eukaryotic expression plasmids of PRV early protein EP0 and Spin1 were respectively constructed and identified correctly by PCR and sequencing,and then co-transfected into human embryonic kidney cells(HEK 293FT).The western blot results showed that the expression level of Spin1 was significantly down-regulated in the cells co-transfected with Spin1 and EP0 expression plasmids,compared with the cells transfected with Spin1 only.To further reveal the mechanism of Spin1 downregulation by EP0,the interaction between EP0and Spin1 was analyzed by co-immunoprecipitation assays(Co-IP).The results showed that EP0 could interact with Spin1 in co-transfected cells.Together,these results suggest that PRV EP0 can down-regulate Spin1 through their interaction.In his study,we identified a new host protein that inhibits PRV replication and elucidated a new mechanism of PRV antagonizing host antiviral factors,providing a theoretical reference for further revealing the pathogenesis of PRV and the development of new drug.