Effect of human aminopeptidase N (hAPN) on porcine deltacoronavirus infecting HEK293 cells

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.

Susceptibility of cell lines from different animal species to Porcine Deltacoronavirus

[Objective] To explore whether porcine deltacoronavirus (PDCoV) can infect and proliferate in different animal species-derived cell lines. [Methods] The Sichuan isolate CHN-SC2015of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey, human and swine. After at least five blindly passages in each cell line, the virus was identified by RT-PCR,RT-q PCR, indirect immunofluorescence assay (IFA), and sequencing. [Results] PDCoV caused distinct cytopathic effect (CPE) in Vero,PAM,PK15,ST, and LLC-PK1 cells at the 1st passage (P1) and proliferated to various degrees in PAM,PK15,ST, and LLC-PK1 cells, while the CPE gradually disappeared during subsequent passages in Vero and PAM cells. Except that in the three susceptible cell lines (PK15,LLC-PK1, and ST), the viral copies of the infected cell lines gradually decreased with the increase in passages, and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02, and PAM cells. PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5. The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02, and specific immunofluorescence was observed in PK15,LLC-PK1, and ST cells at P1,P3, and P9. Therefore, only three cell lines (PK15,LLC-PK1, and ST) were suitable for serial passage, with the virus titers up to 107.11,107.00, and 107.37 TCID50/mL at P9,respectively. After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations. [Conclusion] This study indicates that PDCoV can infect a variety of cells in vitro, suggesting that it may have the potential of cross-species transmission.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.