siRNA干扰马赛病毒潜在翻译起始因子ORF314显著抑制病毒复制

Inhibiting expression of potential translation initiation factor ORF314 with siRNA significantly reduced replication of Marseillevirus in amoeba cells

马赛病毒是继拟菌病毒之后被发现的第2类阿米巴巨型病毒,拥有庞大的双链DNA基因组。基因组分析显示,马赛病毒上海株(Mar-SH2016)编码4种潜在翻译因子。其中,ORF314基因与真核翻译起始因子eIF2/eIF5的氨基酸同源性约为60%,但其在马赛病毒复制中的作用还有待揭示。本研究设计了3对靶向Mar-SH2016 ORF314基因的siRNA,通过转染宿主卡氏棘阿米巴细胞,分析siRNA对ORF314基因表达水平和病毒复制的影响。用荧光标记的siRNA转染卡氏棘阿米巴细胞,结果显示约1/3阿米巴细胞成功转染。实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和蛋白免疫印迹实验证实,在Mar-SH2016感染宿主的过程中siRNA能使ORF314基因表达水平显著下降。siRNA干扰后病毒毒力测定结果显示,Mar-SH2016的毒力降低约30%(P<0.01)。结果表明,降低ORF314基因的表达水平能显著抑制Mar-SH2016复制。

Marseillevirus is a type of giant virus that infects Acanthamoeba castellanii.Like other giant viruses,Marseillevirus has a large genome and encodes several potential protein translation factors.Genomic analysis revealed that Marseillevirus Shanghai strain(Mar-SH2016)encodes four potential translation factors,one of which,ORF314 gene,has a high homology(60%)with the eukaryotic translation initiation factor eIF2/eIF5.But the role of ORF314 in virus replication remains to be revealed.In this study,three pairs of small interfering RNA(siRNAs)were designed to target ORF314 gene.Transfection of amoeba cells with fluorescence-labeled siRNAs showed that about one third of the Acanthamoeba castellanii cells were successfully transfected.Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting confirmed that the siRNAs decreased the mRNA level of ORF314,and the results of virus titration showed that the level of virus replication decreased significantly(P<0.01).It is indicated that the potential translation initiation factor ORF314 of Mar-SH2016 has a role in the replication of the virus.