Construction and Functional Characterization of Stable Cell Lines Expressing Nucleocapsid Protein of Porcine Deltacoronavirus

doi:10.13560/j.cnki.biotech.bull.1985.2025-1397

Abstract

Abstract:

Objective This study aims to establish monoclonal cell lines stably expressing the nucleocapsid (N) protein of porcine deltacoronavirus (PDCoV), which will provide a cellular tool to elucidate its pathogenic mechanism in modulating the host cellular microenvironment to enhance viral proliferation in the viral life cycle. Method Three representative PDCoV strains from different lineages were selected based on phylogenetic analysis, and eukaryotic lentiviral expression plasmids carrying the N gene were constructed. The lentiviral system combined with the limiting dilution method was then used to generate LLC-PK monoclonal cell lines stably expressing PDCoV N protein. Finally, the interferon-β (IFN-β) promoter luciferase reporter system, quantitative real-time PCR (RT-qPCR), flow cytometry, and viral proliferation assays were employed to analyze the effects of stable N protein expression on the type I interferon (IFN-I) pathway, host cell cycle, and viral proliferation ability. Result A total of 30 LLC-PK monoclonal cell lines stably expressing PDCoV N protein were successfully established, including 12 lines of the North American lineage (HT/h0256, a human-derived strain), 9 lines of the Chinese lineage (HT/h0329, a human-derived strain), and 9 lines of the Southeast Asian lineage (THA/S5011). Functional investigation revealed that the stable expression of PDCoV N protein significantly suppresses Sendai virus (SeV)-induced activation of the IFN-β promoter activity and the expression of downstream interferon-stimulated genes (ISG56, ISG15 and MX1), induces pronounced S-phase arrest in LLC-PK cells, and markedly enhances viral proliferation within host cells. Conclusion Stable LLC-PK monoclonal cell lines expressing the N protein of representative strains across three PDCoV lineages were successfully established. The sustained expression of N protein in these cell lines effectively antagonizes the IFN-I signaling pathway, induces host cell S-phase arrest, and enhances viral proliferation, thereby providing a critical cellular model for further dissection of the pathogenic mechanisms underlying N protein-mediated modulation of the host cellular microenvironment.

Key words: porcine deltacoronavirus, nucleocapsid protein, stable cell line, type I interferon pathway, S-phase arrest, virus replication

YANG Tai-ran, ZHENG Xin-qi, HU Chao, AN Ting-ting, WEN Long, LI You-li, CHEN Zheng-hong, CUI Gu-zhen, LI Bo-yan, TIAN Yi-ming. Construction and Functional Characterization of Stable Cell Lines Expressing Nucleocapsid Protein of Porcine Deltacoronavirus[J]. Biotechnology Bulletin, 2026, 42(10): 328-338.

Figures/Tables 5

Fig. 1 Functional domain and amino acid levels analysis of N protein from three PDCoV lineagesA: Phylogenetic genotyping based on 194 complete-genome sequences of PDCoV (red indicates representative strains of PDCoV from three lineages). B: Schematic diagram of PDCoV N protein domain (Green: NTD. Cyan: CTD. Yellow: SR-rich motif). C: Amino acid sequence alignment of N proteins from three PDCoV lineages

Fig. 2 Construction and identification of stable cell lines expressing the N protein from three PDCoV lineagesA: Indirect immunofluorescence assay for detecting the expression and localization of PDCoV N protein from three lineages in polyclonal cell lines (scale bar: 50 μm). B: Western blot analysis of N protein expression levels in monoclonal stable cell lines of three PDCoV lineages (M: LLC-PK control cells). C: Indirect immunofluorescence assay for detecting the expression of N protein in monoclonal stable cell lines of three PDCoV lineages (scale bar: 50 μm). D: CCK-8 assay for evaluating the proliferative activity of stable cell lines of three PDCoV lineages; the ordinate represents cell viability, and the abscissa represents detection time points. *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. No asterisk indicates no statistically significant difference between groups. The same below

Fig. 3 PDCoV N protein suppresses IFN-β activation and downstream stimulated gene expressionA‒C: Analysis of the effect of N proteins from three PDCoV lineages on SeV-induced IFN-β promoter activity using a dual-luciferase reporter assay system (MOCK: Mock control). D‒G: Detection of the relative mRNA expression levels of SeV-induced IFN-β1 (D), ISG56 (E), ISG15 (F) and MX1 (G) genes in stable cell lines of different PDCoV lineages by RT-qPCR. H: Fluorescence microscopy observation of the effect of PDCoV N protein on VSV-GFP replication (green fluorescence intensity indicates VSV replication level. scale bar: 100 μm)

Fig. 4 PDCoV N protein induces S-phase arrest in LLC-PK cellsA: Representative cell cycle profiles of wild-type LLC-PK cells and PDCoV N protein stably transfected cell lines detected by flow cytometry at different time points (0, 12 and 24 h) after serum starvation synchronization (Blue: G0/G1 phase. Brown: S phase. Green: G2/M phase). B: Statistical results of the proportions of cells in G0/G1, S and G2/M phases at different time points

Fig. 5 PDCoV N protein enhances PDCoV replication and proliferationA: Replication kinetics of PDCoV in supernatants of N protein stable cell lines from three lineages using absolute quantitative qPCR. B: Immunofluorescence detection of PDCoV proliferation in stable cell lines expressing the N protein using an anti-spike monoclonal antibody (scale bar: 50 μm)

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