Acta Veterinaria et Zootechnica Sinica ›› 2022, Vol. 53 ›› Issue (5): 1587-1597.doi: 10.11843/j.issn.0366-6964.2022.05.026

• BASIC VETERINARY MEDICINE • Previous Articles     Next Articles

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 Sanjie1,2,3, HUANG Xiaobo1,2,3*   

  1. 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

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

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