A novel host restriction factor MRPS6 mediates the inhibition of PDCoV infection in HIEC-6 cells

Abstract

Introduction: Porcine deltacoronavirus (PDCoV) is a zoonotic pathogen with a global distribution, capable of infecting both pigs and humans. To mitigate the risk of cross-species transmission and potential outbreaks, it is crucial to characterize novel antiviral genes, particularly those from human hosts.

Methods: This research used HIEC-6 to investigate PDCoV infection. HIEC-6 cells were infected with PDCoV. Samples were collected 48 h postinfection for proteomic analysis.

Results: We discovered differential expression of MRPS6 gene at 48 h postinfection with PDCoV in HIEC-6 cells. The gene expression initially increased but then decreased. To further explore the role of MRPS6 in PDCoV infection, we conducted experiments involving the overexpression and knockdown of this gene in HIEC-6 and Caco2 cells, respectively. Our findings revealed that overexpression of MRPS6 significantly inhibited PDCoV infection in HIEC-6 cells, while knockdown of MRPS6 in Caco2 cells led to a significant increase of virus titer. Furthermore, we investigated the correlation between PDCoV infection and the expression of MRPS6. Subsequent investigations demonstrated that MRPS6 exerted an augmentative effect on the production of IFN-β through interferon pathway activation, consequently impeding the progression of PDCoV infection in cellular systems. In conclusion, this study utilized proteomic analysis to investigate the differential protein expression in PDCoV-infected HIEC-6 cells, providing evidence for the first time that the MRPS6 gene plays a restrictive role in PDCoV virus infection.

Discussion: Our findings initially provide the validation of MRPS6 as an upstream component of IFN-β pathway, in the promotion of IRF3, IRF7, STAT1, STAT2 and IFN-β production of HIEC-6 via dual-activation from interferon pathway.

Keywords: IFN-β; MRPS6; PDCoV; host restriction factor; proteomics.

Figure 1

PDCoV infection modeling. We change it according to your suggestions: Figure 1 PDCoV infection modeling. (A) Western blot assay of PDCoV (OK546242) infected different kinds of cells with PDCoV N as the primary antibody. (B) Changes in PDCoV N protein at 12h, 24h, 36h, and 48h in PDCoV-infected HIEC-6 cells. (C) One-step growth curve of PDCoV in HIEC-6 cells, (D) Relative quantification analysis of PDCoV N in HIEC-6 cells infected with PDCoV, Statistical significance is determined by one-way ANOVA ( **P<0.01; ****P<0.0001).

Figure 2

Proteomics analysis of all DEGs. (A) Flow of PDCoV-infected HIEC-6 cell proteomics experiments. (B) Number of DEGs at 48h. (C) Enrichment of DEGs heatmap at 48h. (D) GO annotation of differentially expressed genes or DEGs at 48h. (E) KEGG annotation of differentially expressed DEGs at 48h.

Figure 3

Differential Expression of MRPS6 in PDCoV-infected HIEC-6 cells. (A) Volcano map screening for DEGs of MRPS6. (B) mRNA changes of MRPS6 in HIEC-6 cells infected with PDCoV. (C) Changes of MRPS6 in HIEC-6 cells infected with PDCoV by western blot. (D) MRPS6 gene expression in HIEC-6 and Caco2 by western blot. (E) HIEC-6 cell lesion caused by PDCoV infection. (F) Caco2 cell lesions caused by PDCoV infection, Red arrow positions are cytopathic. Statistical significance is determined by one-way ANOVA (**P<0.01; n.s., not significant).

Figure 4

Overexpression of MRPS6 in HIEC-6 cells mediates the resistance of PDCoV infection. (A) Viral titers and copies are separately detected with RT-qPCR at 12 h and 24 h. (B) Viral titers and copies are separately detected with TCID₅₀. (C) Immunoblotting analysis of PDCoV N indicates the infection level with or without MRPS6 overexpression at 12h and 24h. (D–E) Flow cytometric and immunofluorescence assay were utilized to evaluate the infection at 24 h with PDCoV N antibody as the primary antibody and Cy3-labeled goat anti-rabbit immunoglobulin (Cy3). Statistical significance is determined by one-way ANOVA (**P<0.01; ***P<0.001; ****P<0.0001).

Figure 5

Knockdown of MRPS6 in Caco2 cells mediates the resistance of PDCoV infection. (A) Viral titers and copies are separately detected with RT-qPCR at 12 h and 24 h. (B) Viral titers and copies are separately detected with TCID₅₀. (C) Immunoblotting analysis of PDCoV N indicates the infection level with or without Knockdown of MRPS6 at 12h and 24h. (D–E) Flow cytometric and immunofluorescence assay were utilized to evaluate the infection at 24h with PDCoV N antibody as the primary antibody and Cy3-labeled goat anti-rabbit immunoglobulin (Cy3). Statistical significance is determined by one-way ANOVA (**P<0.01; ****P<0.0001).

Figure 6

MRPS6 plasmid overexpression in HIEC-6 cells at 1 μg, 2 μg, and 4 μg following PDCoV infection using 1 MOI, MRPS6 plasmid overexpression in HIEC-6 cells at 2 μg following PDCoV viral infection using 0.1 MOI, 1 MOI, and 2MOI. (A) PDCoV infection using 1 MOI following MRPS6 plasmid overexpression at 1 μg, 2 μg and 4 μg, 24h immunoblotting assay of PDCoV N antibody as primary antibody (B) RT-qPCR assay of MRPS6 plasmid overexpression of 1 μg, 2 μg and 4 μg followed by PDCoV infection at 1 MOI, (C) PDCoV viral infection using MRPS6 plasmid overexpression of 2 μg in HIEC-6 cells with PDCoV N antibody at 0.1 MOI, 1 MOI and 2MOI, 24h immunoblotting assay of MRPS6 plasmid overexpression of 2 μg as primary antibody. PDCoV N antibody was used as a primary antibody in a 24-hour immunoblotting assay, (D) RT-qPCR assay of MRPS6 plasmid overexpressed in HIEC-6 cells for 2 μg and then infected with PDCoV virus using 0.1 MOI, 1 MOI, and 2MOI. Statistical significance is determined by one-way ANOVA (**P<0.01; ****P<0.0001).

Figure 7

Analysis of MRPS6 interactions and speculation on MRPS6 antiviral pathway. (A) Proteins with significant Interaction with MRPS6. (B) Heatmap analysis of the expression of MRPS6-interacting proteins. (C) MRPS6 was overexpressed in HIEC-6 cells and infected with 1 MOI of PDCoV for 6h, and IFN-β mRNA levels were detected by RT-qPCR. (D) MRPS6 was knocked down in Caco2 cells and infected with 1 MOI of PDCoV for 6h, and IFN-β mRNA levels were detected by RT-qPCR. (E) Poly-IC stimulates activation of IFN pathway and STAT pathway in CaCo2 cells. (F) Overexpression of MRPS6 promotes activation of the IFN-β pathway with the JAK-STAT pathway at different times. (G–H) Western blot and RT-qPCR analysis showed the protein and mRNA levels of IRF-3 after 24h of transfection of the MRPS6 overexpression in HIEC-6 cells. (I–J) Western blot and qRT-PCR analysis revealed the protein and mRNA levels of IRF-3 after 24h of siRNA transfection in Caco2 cells. Statistical significance is determined by one-way ANOVA and T-test (**P<0.01; ***P<0.001; n.s., not significant).

Figure 8

A schematic diagram of MRPS6 involvement in the PDCoV infection and interferon β pathways in the HIEC-6 cell line. Both PDCoV infection and poly-IC induce MRPS6 expression. Upregulated MRPS6 synergistically with the nucleic SNF8 induces IRF3, IRF7, STAT1/2, and IFN β protein production which are the main components of the IFN pathway. The next generation of IFN β would give a second wave of stimuli to strengthen interferon responses and produce more interferon stimulated genes.