HIEC-6, MA104, HEK293, HEK293T, DF1, MDCK, BHK21, ST, LLC-PK1, IPEC-J2, and Caco2 were kept in our laboratory(The cell lines present in this study were obtained from ATCC). All cells were cultured using Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Hyclone). PDCoV (Genbank No. OK546242) was a kind gift from Prof. Bin Li (33).

HRP-conjugated goat anti-rabbit IgG (H+L) (CAT#A0208), Cy3-labeled goat anti-rabbit immunoglobulin (H+L) (Cat #A0516) and FITC-labeled goat anti-mouse immunoglobulin (H+L) (CAT#A0562) were purchased from Beyotime Biotech Inc. Lipo™ RNAiMAX and LIpo™ 3000 were purchased from Invitrogen. SYBR-Green (Promega) and Poly-IC (Merck, USA) were used in the experiments. Anti-PDCoV N rabbit polyclonal antibody was prepared in our laboratory. Specific antibodies for MRPS6 and GAPDH were separately purchased from ORIGENE and Genetex. Polyclonal antibody for IRF-3 was purchased from Sanying (Proteintech). Antibody for STAT1, STAT2, IRF7 and IFN-Beta was purchased from Cell Signaling Technology.

pCAGGS-MRPS6 was constructed in our laboratory. Briefly, MRPS6 was obtained by amplification using primers and pCAGGS-MRPS6 was obtained by subcloning EcoR I and Not I into pCAGGS (Inovogen Technologies). Recombinant plasmids were validated by PCR and sequencing.

LLC-PK1 cells were inoculated with PDCoV for virus replication. The virus was subsequently diluted with DMEM containing 10 μg/mL trypsin. After incubation at 37°C for 2 hours, the medium of host cell LLC-PK1 was changed to remove unattached virus until 50% cell were induced to produce cytopathic effect (CPE). The cells were then subjected to one cycle of freeze-thawing, and the supernatant containing the virus was collected by centrifugation at 12,000 rpm for 1 min.

HIEC-6 cells were seeded into 12-well plates at a concentration of 2 × 10⁵ cells per well, incubated overnight to reach 70%–80% confluency, and infected with the PDCoV virus at a MOI of 1. The cell-virus admixture was harvested at time intervals of 6, 12, 24, 36, 48 and 60 hours postinfection. Each collected sample underwent a single freeze-thaw cycle, followed by centrifugation at 12,000 rpm for 1 min to precipitate cellular debris, thereby allowing for the isolation of the viral supernatant.

We have determined the virus titer as previous description (34). Briefly, HIEC-6 or Caco2 cells were seeded in 96-well plates at a density of 1×10⁵ cells per well. The PDCoV samples were then diluted serially at a 10-fold dilution with 100 µL per well, and each sample was repeated three times. The TCID₅₀ (median tissue culture infectious dose) was subsequently calculated using the Reed-Muench method.

The coding sequence of human MRPS6 (NM_032476.4) was amplified from Caco2 cells and subsequently inserted into the pCAGGS vector. HIEC-6 cells were cultured until reaching 80% confluency and transfected with the pCAGGS-MRPS6 plasmid or poly-IC (60 mM per well) using LIpo™ 3000 Transfection Reagent. MRPS6 siRNA(stB0013147A) purchased from RiboBio was transfected into Caco2 with 80–90% confluency with RNAiMAX transfection reagent for preparation.

The protein samples were subjected to extraction and subsequently underwent quality control. Those samples that passed the quality control were then separated and subjected to liquid quality control in DDA mode. The obtained data from DDA scanning mode were searched against the Human Protein Data Bank using Spectronaut-Pulsar (version 14.0, Biognosys), a library search software. Once the search was completed, Spectronaut software (version 14.0, Biognosys) was utilized to directly generate spectral libraries. Additionally, interscan software was employed to annotate the GO functions, which included Pfam, PRINTS, ProDom, SMART, ProSite, and PANTHER databases. Functional protein family, and pathway analysis of identified proteins by KEGG (35). Volcano plot analysis, clustered heatmap analysis, and pathway enrichment analysis for DEGs (DEGs) were conducted. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were utilized for pathway enrichment analysis. Additionally, potential protein-protein interactions (PPI) were predicted using the STRING DB software (http://STRING.embl.de/) (36).

Cells were collected and lysed, and protein concentrations were determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Cat#P0010). The proteins were mixed with loading buffer and denatured by boiling. Then, equal amounts (30 μg) were electrophoresed and transferred onto a nitrocellulose filter membrane, and then incubated overnight at 4°C with primary antibodies. After further incubation with HRP-conjugated secondary antibodies, the membranes were detected by the Amersham Imaging 600 system, with Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Cat#32106) (37).

Total cellular RNA was extracted and quantified following the manufacturer's instructions (Sangon Biotech, Cat# B511311). Total RNA was employed as a template and subjected to reverse transcription using M-MLV reverse transcriptase (Promega) to generate complementary DNA (cDNA). The cDNA was subsequently subjected to a real-time quantitative polymerase chain reaction (RT-qPCR) using SYBR Green Master Mix mixed with primers from ( Table 1 ) on a BIO-RAD CFX96 RT-qPCR system (BIO-RAD). The genes were then quantified using quantitative real-time PCR and analyzed using the 2-∆∆CT method (38).

HIEC-6 or Caco2 cell lines were seeded into 12-well plates at a density of 5 × 10⁵ cells per well. The target plasmids were transfected and cultured for 36h. Subsequently, the cells were infected with the PDCoV virus at a MOI of 1 and cultured for an additional 24 hours. Following this, the cells were fixed with 4% paraformaldehyde for 30 minutes and washed thrice with PBS. The cells were then permeabilized with 0.1% Triton X-100 and blocked with 5% skimmed milk for 1 hour. Subsequently, the cells were incubated with FITC-coupled PDCoV N protein-specific primary antibody for 2 hours at 37°C. After washing with PBS, fluorescence photography was conducted.

Samples were collected from different host cells at 24 hours postinfection with 1 MOI PDCoV. The cells were enzymatically digested using trypsin and subsequently resuspended. The cells were then permeabilized with 0.1% Triton X-100 and blocked with 5% skim milk for 1 hour. Subsequently, the cells were incubated with FITC-coupled PDCoV N protein-specific primary antibody for 2 hours at 37°C. After washing with PBS, fluorescence photography was conducted and analyzed by CytoFLEX flow cytometry.

Statistical significance between groups was determined using GraphPad Prism, version 9.3.1 Data were presented as means ± standard errors of the means (SEM) in all experiments and analyzed using a one-way ANOVA and T-test, and a P value of < 0.05 was considered to be statistically significant.

We evaluated the infection range of PDCoV by inoculating them into ten different cell lines (HIEC-6, MA104, HEK293, HEK293T, DF1, MDCK, BHK21, ST, IPEC-J2, and Caco2). The PDCoV N was detected using a western blot to prove the existence and infection of the virus. The specific 42kDa bands were shown in all the cells. However, we discovered a higher expression from the result of avian-derived DF1, African green monkey embryonic kidney cells derived MA104, porcine-derived IPEC-J2, and human-derived Caco2 or HIEC-6 ( Figure 1A ). Subsequently, we opted for the utilization of HIEC-6 cells, which exhibited a greater level of infection, for subsequent experiments. We conducted an evaluation of the expression of PDCoV N protein at various time points (12h, 24h, 36h, and 48h). Immunoblot analysis revealed the presence of the target strip measuring 42kDa at all time intervals, with the maximum intensity observed at 24h. Subsequently, no significant changes in protein expression were observed ( Figure 1B ). The viral titer was assessed, with the highest level observed at 36h, reaching 10^5.5 TCID₅₀/0.1ml ( Figure 1C ). To quantify the expression levels of PDCoV N, RT-qPCR was performed with GAPDH as the internal reference. The mRNA expression level PDCoV N expression level showed a significant difference at 48h ( Figure 1D ). In conclusion, PDCoV can infect HIEC-6 cells and replicate within HIEC-6.

The objective of this study was to examine the impact of PDCoV infection on HIEC-6 cells after 48 hours by evaluating alterations in gene expression induced by the virus. Uninfected cells were utilized as a control group (referred to as “Mock”). The 4D-DIA technique was employed to analyze the samples ( Figure 2A ). A total of 243 DEGs were identified at 48h, with 93 DEGs being upregulated and 150 DEGs being downregulated ( Figures 2B, C and Supplementary Tables 1, 2 ). Gene ontology (GO) enrichment analysis demonstrated that the enriched biological processes were associated with mRNA polyadenylation, RNA processing, RNA methyltransferase activity, and other related processes ( Figure 2D ). Furthermore, KEGG pathway analysis revealed enrichment of pathways linked to the Rap1 signaling pathway, cytokine-cytokine receptor interaction, and cancer ( Figure 2E ).

Significant differential expression of the MRPS6 gene was observed at 48 hours in PDCoV-infected HIEC-6 cells compared to uninfected cells, as determined by statistical analysis (P < 0.05) and a log2 fold change greater than 1.5. This finding was obtained through a comparative analysis of the volcanic plots, where the top 20 differentially expressed genes were identified and presented in Figure 3A . PDCoV-infected HIEC-6 cells were collected at different time points (12, 24, 36, and 48 hours) to investigate the expression levels of MRPS6 using RT-qPCR ( Figure 3B ). Western blot analysis ( Figure 3C ) confirmed the initial increase and subsequent decrease in MRPS6 expression from 24 to 48 hours. The highest expression of MRPS6 was observed at 24 hours of PDCoV infection. To compare MRPS6 expression between HIEC-6 and Caco2 cells, protein validation was performed, revealing significantly higher levels of MRPS6 in Caco2 cells compared to HIEC-6 cells ( Figure 3D ). Morphological analysis of PDCoV-infected HIEC-6 and Caco2 cells at 48h showed floating cells and vacuolated lesions, respectively ( Figures 3E, F ). At 48 hours post-infection, HIEC-6 cells infected with PDCoV exhibited a floating phenotype, while Caco2 cells displayed vacuolated lesions. These observations lay the groundwork for our subsequent investigations, which aim to elucidate the impact of PDCoV infection on the expression of MRPS6, evaluate the effects of MRPS6 overexpression in the HIEC-6 cell line, conduct knockdown experiments in the Caco2 cell line, and determine the TCID₅₀ based on the observed cellular lesions.

HIEC-6 cells were utilized to investigate the overexpression of MRPS6 using the pCAGGS vector. After 36 hours of overexpression, the cells were infected with a virus at a MOI of 1. To assess the functional impact of MRPS6 overexpression, multiple experimental techniques were utilized, including Western blot analysis, RT-qPCR, TCID₅₀ assay, flow cytometry, and immunofluorescence. The primary antibody employed in this study was PDCoV N, which specifically detected a band at 42kDa. The results obtained from the Western blot analysis demonstrated a significant decrease in the expression of the target band, as well as the N protein, following the overexpression of MRPS6 ( Figure 4A ). Additionally, the TCID₅₀ assay revealed a reduction in viral titer in the supernatant at both 12h and 24h post-infection ( Figure 4B ). mRNA detection exhibited a notable disparity in N protein expression at both time points ( Figure 4C ). Flow cytometry analysis, utilizing FITC-coupled anti-PDCoV N antibody, exhibited a lower proportion of positive cells in HIEC-6 cells overexpressing MRPS6 in comparison to the plasmid-transfected group ( Figure 4D ). Finally, immunofluorescence results, utilizing PDCoV N as the primary antibody, indicated a significantly diminished specific fluorescence of N protein in cells overexpressing MRPS6 compared to non-transfected cells ( Figure 4E ). These findings suggest that the overexpression of MRPS6 can effectively reduce PDCoV virus infection.

Subsequently To elucidate the functional role of MRPS6, a variety of experimental techniques were employed, including Western blot analysis, RT-qPCR, TCID₅₀, flow cytometry, and immunofluorescence. The primary antibody utilized in this investigation specifically targeted the PDCoV N protein, enabling the detection of a band at a molecular weight of 42 kDa. The results obtained from the Western blot analysis revealed an upregulation in the expression of the PDCoV N protein following MRPS6 inhibition ( Figure 5A ). Additionally, the TCID₅₀ assay demonstrated an increase in the viral titer in the supernatant at both 12h and 24h post-infection ( Figure 5B ). RT-qPCR analysis also exhibited a significant disparity in the expression of the N protein at both time points ( Figure 5C ). Flow cytometry analysis, utilizing a FITC-conjugated anti-PDCoV N antibody, displayed a higher percentage of positive cells in Caco2 cells with MRPS6 inhibition compared to the siRNA-NC group ( Figure 5D ). Finally, the immunofluorescence results, utilizing the PDCoV N protein as the primary antibody, revealed significantly higher specific fluorescence of N proteins in MRPS6-inhibited cells as opposed to untransfected cells ( Figure 5E ). These findings strongly indicate that MRPS6 inhibition effectively enhances PDCoV infection.

Investigations were conducted to assess the inhibitory effects of various concentrations of MRPS6 on PDCoV infection in HIEC-6 cells. The cells were transfected with either 1 μg, 2 μg, or 4 μg of MRPS6 and subsequently infected with PDCoV. The effects were determined using Western blot and RT-qPCR techniques. Our findings demonstrated that the overexpression of 4 μg of MRPS6 significantly inhibited PDCoV viruses to a greater extent compared to the overexpression of 1 μg and 2 μg of MRPS6 ( Figures 6A, B ). The inhibitory efficacy of MRPS6 on Porcine Deltacoronavirus (PDCoV) exhibited a dose-dependent relationship, with higher concentrations demonstrating a significantly greater inhibitory effect compared to lower concentrations. To investigate the potential impact of MRPS6 on the infection of PDCoV at different MOIs, HIEC-6 cells were transfected with 4 μg of MRPS6 and subsequently infected with PDCoV at MOIs of 0.1, 1, and 2. The infection was evaluated using Western blot and RT-qPCR techniques. The results demonstrated that the overexpression of 4 μg of MRPS6 had a significantly stronger inhibitory effect on PDCoV at an MOI of 0.1 compared to an MOI of 1 and 2 ( Figures 6C, D ). These findings suggest that MRPS6 exerts a more potent inhibitory effect on PDCoV at lower MOI values.

Identification of MRPS6-interacting proteins was conducted using proteomic data from HIEC-6 cells infected with PDCoV. A total of eight genes were found ( Figure 7A ), with SNF8, HIST1H1D, and MRPS18A showing up-regulation, while RPS16, POLR2J, TARBP1, and RPL4 showed down-regulation ( Figure 7B ). We examined whether MRPS6 exerts regulatory effects on IFN-β in HIEC-6 cells. Following MRPS6 overexpression and PDCoV inoculation, RT-qPCR analysis revealed that MRPS6 overexpression promoted IFN-β transcription ( Figure 7C ). Subsequently, we conducted knockdown experiments on MRPS6 in Caco2 cells, which were then infected with 1MOI PDCoV. The mRNA level of IFN-β was evaluated using RT-qPCR 6 hours later. The results demonstrated a significant reduction in the mRNA level of IFN-β upon MRPS6 knockdown ( Figure 7D ). Our findings also prove that MRPS6 in the HIEC-6 cell could be induced by poly-IC, and other main components of IFN-β pathway, such as STAT1, STAT2, IRF7 and even IFN-β itself, was upregulated due to the activation role ( Figure 7E ). Further data revealed maximal expression level of MRPS6 was at 24 hours post-transfection. The expression of IFN-β, STAT1, STAT2, and IRF7 was strongly correlated with MRPS6 overexpression ( Figure 7F ). It indicates that MRPS6 can activate various downstream components from IFN-β pathway. Considering that IRF-3 is the primary transcription factor for IFN-β [38], we investigated the impact of MRPS6 on IRF-3 in HIEC-6 cell. Overexpression of MRPS6 in HIEC-6 cells resulted in increased protein and mRNA levels of IRF-3 ( Figures 7G, H ). Furthermore, we performed knockdown and overexpression experiments on MRPS6 in Caco2 cells and assessed the expression levels of IRF-3 using WB and RT-qPCR. The findings revealed a significant reduction in both the protein and mRNA levels of IRF-3 upon MRPS6 knockdown in Caco2 cells ( Figures 7I, J ).

Additionally, a previous study has demonstrated that RPS16, an MRPS6-interacting protein, plays a role in inhibiting viral replication and increasing the expression of type I interferon when its expression is knocked down (39). SNF8 has been demonstrated to interact with IRF3 and CBP to enhance the activation of the interferon antiviral response. This suggests that SNF8 is necessary for the optimal induction of the IRF3-dependent innate antiviral defense mechanism (40). Previous studies have postulated that HCV inhibits the translation of ISG proteins at the ribosome and confines viral replication to cellular compartments that are not susceptible to antiviral IFN-stimulated effector systems (41).

In this investigation, we believe that PDCoV infected HIEC-6 cells internalize the virus through receptor-mediated endocytosis. Viral RNA translocates to the Golgi apparatus in the presence of RPS16, a protein known to interact with MRPS6. From the Golgi, viral RNA is subsequently transported to the mitochondria, where MRPS6, a subunit of the mitochondrial ribosomal protein, facilitates the translation of viral proteins. Because poly-IC stimulation to HIEC-6 cell leads to increased MRPS6 expression and an associated upregulation of the interferon-β pathway, we confirmed that MRPS6 is involved in the interferon-β pathway. Overexpression of MRPS6 further amplifies IRF-3 and IRF-7 activation, which enhances IFN-β production. The increase in IFN-β levels consequently reactivates their receptors, culminating in the upregulation of STAT1 and STAT2. Thus, this is a dual activation of interferon pathway coupled with both IRFs and JAK-STAT pathway, which present an obvious anti-PDCoV mechanism shown as Figure 8 .