人氨基肽酶N在猪丁型冠状病毒感染HEK293细胞中的作用

doi:10.11843/j.issn.0366-6964.2022.05.026

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (5): 1587-1597.doi: 10.11843/j.issn.0366-6964.2022.05.026

人氨基肽酶N在猪丁型冠状病毒感染HEK293细胞中的作用

赵玉佳¹, 陈汭¹, 宋代丽¹, 张路文¹, 肖黛¹, 李施倩¹, 文翼平¹, 伍锐¹, 赵勤¹, 杜森焱¹, 颜其贵¹, 文心田¹, 曹三杰^1,2,3, 黄小波^1,2,3*

1. 四川农业大学动物医学院猪病研究中心, 成都 611130;
2. 农业农村部兽用药物与兽医诊断技术四川科学观测实验站, 成都 611130;
3. 四川农业大学国家级动物类实验教学示范中心, 成都 611130

收稿日期:2021-09-03 出版日期:2022-05-23 发布日期:2022-05-25
通讯作者: 黄小波,主要从事预防兽医学研究,E-mail:rsghb110@126.com
作者简介:赵玉佳(1989-),女,河南新乡人,博士生,主要从事动物传染病研究,E-mail:zhaoyujia2015@163.com
基金资助:
国家自然科学基金项目(32172888)；四川省国际科技创新合作项目(2021YFH0153)；云南省重大科技专项计划(202102AE090039)

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

ZHAO Yujia¹, CHEN Rui¹, SONG Daili¹, ZHANG Luwen¹, XIAO Dai¹, LI Shiqian¹, WEN Yiping¹, WU Rui¹, ZHAO Qin¹, DU Senyan¹, YAN Qigui¹, WEN Xintian¹, CAO Sanjie^1,2,3, HUANG Xiaobo^1,2,3*

1. Research Center for Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture and Rural Affairs, Chengdu 611130, China;
3. National Animal Experiments Teaching Demonstration Center, Sichuan Agricultural University, Chengdu 611130, China

Received:2021-09-03 Online:2022-05-23 Published:2022-05-25

摘要/Abstract

摘要： 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TYR⁹²、THR⁵¹、THR⁴⁸、PHE¹⁶和MET¹⁴通过氢键与hAPN蛋白的氨基酸残基PHE⁴⁹⁰、GLN⁵³¹、ARG⁵²⁸和SER⁵²⁹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。

关键词: 猪丁型冠状病毒, 氨基肽酶N, HEK293细胞, 复制

Abstract: 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 TYR⁹², THR⁵¹, THR⁴⁸, PHE¹⁶ and MET¹⁴of S1 protein receptor binding motif 1 (RBM1) can form hydrogen bonds with residues PHE⁴⁹⁰, GLN⁵³¹, ARG⁵²⁸ and SER⁵²⁹ 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.

Key words: porcine deltacoronavirus, aminopeptidase N, HEK293 cells, replication

中图分类号:

S852.659.6

赵玉佳, 陈汭, 宋代丽, 张路文, 肖黛, 李施倩, 文翼平, 伍锐, 赵勤, 杜森焱, 颜其贵, 文心田, 曹三杰, 黄小波. 人氨基肽酶N在猪丁型冠状病毒感染HEK293细胞中的作用[J]. 畜牧兽医学报, 2022, 53(5): 1587-1597.

ZHAO Yujia, CHEN Rui, SONG Daili, ZHANG Luwen, XIAO Dai, LI Shiqian, WEN Yiping, WU Rui, ZHAO Qin, DU Senyan, YAN Qigui, WEN Xintian, CAO Sanjie, HUANG Xiaobo. Effect of Human Aminopeptidase N(hAPN) on Porcine Deltacoronavirus Infecting HEK293 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1587-1597.

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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

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