人氨基肽酶N在猪丁型冠状病毒感染HEK293细胞中的作用

Effect of Human Aminopeptidase N(hAPN)on Porcine Deltacoronavirus Infecting HEK293 Cells

以HEK293细胞为模型,探讨人氨基肽酶N(human aminopeptidase N,hAPN)在猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)入侵人源细胞中的作用。RT-qPCR/RT-PCR鉴定PDCoV在HEK293细胞上的增殖情况,再用CRISPR/Cas9技术构建敲除hAPN基因的HEK293细胞系,通过CCK-8试验验证敲除细胞和野生型细胞的细胞活性;用RT-qPCR和Western blot检测hAPN敲除和过表达对PDCoV复制的影响;进一步通过同源建模和分子对接预测PDCoV S蛋白和hAPN蛋白的结合位点。结果显示:PDCoV在感染HEK293细胞后12~36 h快速增殖,在36 h达到顶峰,在HEK293细胞上至少可传至2代;hAPN敲除细胞与野生型细胞的细胞活性无明显差异,细胞敲除hAPN可显著抑制PDCoV复制,细胞过表达hAPN可促进PDCoV复制;同源建模和分子对接表明,S1蛋白可与hAPN蛋白结构域II结合,主要是S1蛋白的受体结合基序1(receptor binding motif 1,RBM1)氨基酸残基TYR^(92)、THR^(51)、THR^(48)、PHE^(16)和MET^(14)通过氢键与hAPN蛋白的氨基酸残基PHE^(490)、GLN^(531)、ARG^(528)和SER^(529)结合。hAPN在PDCoV感染人源细胞中发挥重要作用,为深入研究PDCoV的细胞入侵和跨种传播机制提供了新的理论依据。

HEK293 cells were used as the cell model to investigate the role of human aminopeptidase N(hAPN)in the invasion of porcine deltacoronavirus(PDCoV)into human cells.The proliferation of PDCoV on HEK293 cells was firstly identified by RT-qPCR/RT-PCR.And then,hAPN knockout cell line was constructed by CRISPR/Cas9 technology and cell viability of HEK293 hAPN knockout and wild-type cells was verified by CCK-8 assay.Effect of hAPN knockout and overexpression on PDCoV replication was detected by RT-qPCR and Western blot.Meanwhile,interaction of PDCoV S protein and hAPN protein was analyzed by homology modeling and molecular docking.Results showed that PDCoV virus copies rapidly increased at 12-36 h and reached peak level at 36 h,it could propagate at least for passage 2 on HEK293 cells.There was no significant difference in cell viability between hAPN knockout cells and wild-type cells.Knockout of hAPN inhibit PDCoV replication and overexpression of hAPN enhance PDCoV replication.Homology modeling and molecular docking analysis showed S1 protein could bind hAPN domain II.Residues TYR^(92),THR^(51),THR^(48),PHE^(16) and MET^(14)of S1 protein receptor binding motif 1(RBM1)can form hydrogen bonds with residues PHE^(490),GLN^(531),ARG^(528) and SER^(529) of hAPN.This study indicates that hAPN plays a critical role in HEK293 cells during PDCoV infection,which provides new theoretical evidence for further studies on the mechanism of PDCoV entry into host cells and cross-species transmission.