Porcine transmissible gastroenteritis virus inhibits NF-κB activity via nonstructural protein 3 to evade host immune system

Intestinal epithelial cell lines J2 (IPEC-J2) and HEK-293 T, were available in our laboratory. The swine testicular (ST) cells were obtained from the American Type Culture Collection (ATCC, CRL-1746). The ST cells and IPEC-J2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 12491015, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10099141, USA) at 37 °C and 5% CO₂. The TGEV TH-98 strain was isolated from the intestinal tract of TGEV-infected piglets in the Heilongjiang province of China (GenBank accession number: KU729220). The virus titer was determined using 50% tissue culture infective dose (TCID 50) assay. Mouse anti-β-actin and mouse anti-hemagglutinin (HA) monoclonal antibodies (mAbs) were purchased from Sigma (A1978, H7411, USA), while mouse mAbs against p65 and IκBα and rabbit mAbs against phospho-NF-κB p65 (6956 T, 4814 T, 3033 T, respectively) were obtained from Cell Signaling Technology (USA). The synthetic double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)) was supplied by Sigma (P9582, USA). A cell viability assay was performed using the Cell Counting Kit-8, following the manufacturer’s instructions (Sangon Biotech, E606335–0100, China).

The eukaryotic expression vectors pCMV-HA and pCMV-Myc were purchased from Clontech (635690 and 635689, respectively, Japan). The NF-κB luciferase reporter plasmid pNF-κB-Luc was supplied by Beyotime Biotechnology (D2206, China). The internal reference gene reporter plasmid pRL-TK was provided by Promega (E2241, USA). The eukaryotic expression plasmids of ubiquitin protein (Ub), TGEV-encoded proteins, and Nsp3 fragments used in this study were constructed in our laboratory. The primers used are shown in Table 1.

For TGEV infection studies, ST or IPEC-J2 cells were seeded in 24-well cell culture plates. When the cells reached a confluency of 70–80%, the cells were co-transfected with pNF-κB-luc (0.5 μg) and reference plasmid pRL-TK (0.025 μg). After 12 h, the cells were treated with poly(I:C) (10 μg/mL) or sterile phosphate-buffered saline (PBS). After 24 h, the cells were infected with TGEV. The infected cells were lysed at 12, 24, and 36 h post-infection. Firefly luciferase and Renilla luciferase activities were determined using a dual luciferase reporter assay system (Promega, USA), following the manufacturer’s instructions. For TGEV gene transfection studies, HEK-293 T or IPEC-J2 cells were seeded in 24-well cell culture plates. When the cells reached a confluency of 70–80%, the cells were co-transfected with pNF-κB-luc, reference plasmid pRL-TK, and either a pCMV-HA expression plasmid containing TGEV genes or an empty pCMV-HA plasmid. After 24 h, the cells were incubated with either poly(I:C) (10 μg/mL) or sterile PBS for 12 h and the cells were collected for dual luciferase activity analysis. All the values were normalized using Renilla luciferase activity as an internal control and expressed in terms of fold change. The data are represented as mean ± standard deviation from three independent experiments.

The cells were washed with PBS and total cellular RNA was extracted using RNA Rapid extraction kit, following the manufacturer’s instructions (Fastagen, 220010). Total RNA was reverse transcribed into cDNA using random primers and M-MLV Reverse Transcriptase (639574, TaKaRa, Japan). The cDNA was used as a template in the SYBR Green PCR assay (Roche, Germany). The abundance of individual mRNA transcripts in each sample was assayed thrice using β-actin as an internal control. Changes in fluorescence signal throughout the reaction were detected in the ABI PRISM 7500 Real-Time PCR system. The relative transcript levels of interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-α were calculated according to the 2^−ΔΔCt threshold method. Primers are listed in Table 2.

The cells were washed with ice-cold PBS and treated with a cell lysis buffer (Beyotime, P0013G, China) containing a protease inhibitor cocktail (Sigma, P8340, USA). Nuclear and cytosolic proteins were isolated with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027, China), following the manufacturer’s instructions. The cells were lysed on ice for 30 min, and the cellular debris was removed by centrifugation. The protein concentration in the lysate was quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime, P0011, China). The protein samples were mixed with 5X sodium dodecyl sulfate (SDS) loading buffer and boiled for 10 min. The samples were subjected to SDS polyacrylamide gel electrophoresis (PAGE) and western blotting to quantify the expression of TGEV total p65, cytoplasm IκBα, cytoplasm p-p65, and nuclear p65 using the respective antibodies. β-actin was used as the loading control.

For co-immunoprecipitation assay, IPEC-J2 and HEK-293 T cells were cultured in 100 mm dishes and transfected with pCMV-Myc-Nsp3 (590–1215 aa) and pCMV-HA-Ub for 24 h. Next, the cells were treated with 10 μg/mL poly(I:C) for 12 h. The cells were harvested and lysed with a cell lysis buffer for performing immunoprecipitation assays. MG132 (25 mM) was added to the culture medium 4 h before harvesting the cells. The samples were incubated on a plate shaker at 4 °C for 30 min. The supernatant was transferred to fresh tubes and incubated with control IgG antibody coated on agarose beads at 4 °C for 2 h. The samples were incubated with Myc monoclonal antibodies coated on agarose beads at 4 °C for 2 h. The mixture was centrifuged at 1,000 rpm and 4 °C for 1 min. The pellets were washed five times with PBS and analyzed by western blotting using HA monoclonal antibodies.

Poly(I:C) is a synthetic analog of double-stranded RNA (dsRNA), which is recognized by the Toll-like Receptor 3 (TLR3). Poly(I:C) activates NF-κB signaling pathway and induces cytokine production [23]. The antiviral effect of poly(I:C) against TGEV was evaluated by treating the IPEC-J2 cells with poly(I:C) for 12 h before inoculation with the TGEV. The RT-PCR analysis revealed that poly(I:C) can significantly reduce the TGEV RNA replication, whereas its effect on cell viability was minimal (Fig. 1a). Although poly(I:C) inhibited TGEV replication in the cells, the virus could not be completely inactivated. Hence, we hypothesized that TGEV may evade the host immune system by inhibiting the poly(I:C)-activated NF-κB pathway. We co-transfected ST and IPEC-J2 cells with the pNF-κB-Luc reporter plasmid to evaluate the effect of TGEV replication on the NF-κB signaling pathway. The pRL-TK plasmid was used as an internal reference. At 12 h post-transfection, the cells were treated with poly(I:C) to induce the activation of NF-κB signaling pathway. At 24 h post-transfection, the cells were infected with TGEV at a multiplicity of infection (MOI) of 1. The infected cells were harvested for dual luciferase activity analysis at different time points. We observed that the poly(I:C)-treated group exhibited significant activation of the NF-κB signaling pathway when compared to the mock control group. However, TGEV infection resulted in time-dependent inhibition of NF-κB signaling activation (Fig. 1b). The transfected ST and IPEC-J2 cells were treated with poly(I:C) and infected with TGEV at different MOIs to evaluate the effect of viral infection titers on the inhibition of NF-κB signaling pathway. As shown in Fig. 1c, NF-κB signaling was significantly activated in the poly(I:C)-treated group when compared to the control group. TGEV infection resulted in significant dose-dependent inhibition of the NF-κB pathway activation.

The role of key TGEV proteins involved in the inhibition of NF-κB signaling pathway was evaluated by transfecting the plasmids encoding TGEV proteins into the HEK-293 T and IPEC-J2 cells. The inhibition of NF-κB signaling pathway was assessed using a luciferase reporter assay system. The luciferase reporter analysis indicated that all TGEV proteins, except Nsp2, inhibited the NF-κB signaling pathway to varying extents. Moreover, Nsp1 and Nsp3 were the most potent inhibitors of NF-κB signaling (Fig. 2a). The degree of NF-κB signaling pathway inhibition in the host cell exerted by Nsp3 was evaluated by transfecting the IPEC-J2 and HEK-293 T cells with increasing doses of Nsp3-expressing plasmids. We observed that Nsp3 could dose-dependently suppress the activation of NF-κB signaling pathway (Fig. 2b). These results indicate that Nsp3 plays an important role in the inhibition of NF-κB signaling pathway during TGEV infection.

NF-κB activation is characterized by the degradation of IκBα as well as the phosphorylation and nuclear translocation of p65 [24]. Therefore, it is important to determine the effect of Nsp3 on IκBα and p65. HEK-293 T (Fig. 3a) and IPEC-J2 (Fig. 3b) cells were transfected with either different titers of Nsp3 or an empty vector. Further, the transfected cells were treated with poly(I:C) for activating NF-κB. The nuclear and cytoplasmic proteins of the cells were extracted and the expression levels of p65, IκBα, and p-p65 were quantified by western blotting. Western blotting analysis revealed that the IκBα expression gradually increased with an increase in the dose of Nsp3-expressing plasmid. Moreover, we observed that Nsp3 had no significant contribution to the total amount of p65. However, the levels of phosphorylated and nuclear p65 decreased with an increase in the Nsp3 levels. These data indicate that Nsp3 inhibits the degradation of IκBα as well as the phosphorylation and nuclear translocation of p65.

Next, we investigated whether TGEV Nsp3 inhibits NF-κB-mediated cytokine production. HEK-293 T and IPEC-J2 cells were transfected with either Nsp3-expressing plasmid or pCMV-HA vector. The cells were treated with poly(I:C) to induce the activation of NF-κB signaling pathway at 24 h post-transfection. The mRNA levels of IL-1, IL-6, IL-8, TNF-α, and β-actin in cells were quantified using RT-PCR at 12 h post-treatment. The expression levels of IL-1, IL-6, IL-8, and TNF-α in the Nsp3-transfected group were lower when compared to those in the poly(I:C) treatment group (Fig. 3b). These findings demonstrate that Nsp3 inhibits NF-κB-regulated cytokine gene expression by inhibiting the NF-κB signaling pathway in both HEK-293 T (Fig. 4a) and IPEC-J2 (Fig. 4b) cells.

The key functional domains of TGEV Nsp3 involved in the inhibition of NF-κB signaling pathway were examined using expression vectors that encoded truncated Nsp3. The truncated expression vectors were constructed based on the structure of TGEV Nsp3, which was predicted by the online program SMART (http://​smart.​embl-heidelberg.​de/​). The IPEC-J2 and HEK-293 T cells were co-transfected with the truncated Nsp3 and the pNF-κB-Luc reporter plasmid. The cells were then treated with poly(I:C) at 24 h post-transcription. Further, luciferase activity and gene expression in the cells were quantified. The luciferase activity analysis revealed that Nsp3 (1–418 aa) and Nsp3 (590–1,215 aa) inhibited the NF-κB signaling pathway activation in both IPEC-J2 and HEK-293 T cells (Fig. 5a). Moreover, RT-PCR analysis indicated that mRNA levels of the NF-κB-related cytokines (IL-1, IL-6, IL-8, and TNF-α) were downregulated upon transfection with Nsp3 (1–418 aa) and Nsp3 (590–1215 aa) plasmids (Fig. 5b). Particularly, Nsp3 (590–1215 aa)-transfected cells exhibited significant attenuation of the NF-κB signaling pathway and expression of NF-κB-regulated cytokines when compared to theother Nsp3 truncated expression vectors(1–418 aa)-transfected cells. The effect of Nsp3 (590–1,215 aa) expression on the inhibition of the NF-κB signaling pathway was examined by co-transfecting pNF-κB-Luc reporter plasmid and different doses of Nsp3 (590–1,215 aa) eukaryotic expression plasmid into the HEK-293 T and IPEC-J2 cells. The Nsp3 (590–1215 aa)-transfected cells exhibited a dose-dependent inhibition of the NF-κB activation (Fig. 5c).

The effect of Nsp3 (590–1,215 aa) transfection on the expression of IκBα and p65, which are key proteins in the NF-κB signaling pathway, was investigated using the HEK-293 T and IPEC-J2 cells co-transfected with Nsp3 (590–1,215 aa) eukaryotic expression recombinant plasmid. The co-transfected cells were treated with poly(I:C) to activate the NF-κB pathway. The nuclear and cytoplasmic proteins were extracted at 36 h post-transfection and the expression levels of p65, IκBα, and p-p65 were quantified by western blotting. We observed a gradual increase in the protein expression level of IκBα with an increase in the Nsp3 (590–1,215 aa) protein expression in both HEK-293 T (Fig. 6a) and IPEC-J2 (Fig. 6b) cells, without affecting the total amount of intracellular p65. However, the level of cytoplasmic p-p65 and nuclear p65 decreased with an increase in the Nsp3 (590–1,215 aa) expression level. These results indicate that Nsp3 (590–1,215 aa) dose-dependently inhibits the degradation of IκBα as well as the phosphorylation and nuclear translocation of p65.

The mechanism underlying the suppressive effects of Nsp3 on the NF-κB signaling pathway was evaluated by co-transfecting the HA-tagged ubiquitin eukaryotic expression plasmid as well as the Myc-tagged Nsp3 and its truncated gene fragments into the IPEC-J2 and HEK-293 T cells. The level of protein ubiquitination levels were quantified by western blotting. The results showed that the high expression of Nsp3, Nsp3 (1–418 aa), and Nsp3 (590–1,215 aa) reduced cellular protein ubiquitination to varying degrees. Moreover, the deubiquitination effect of Nsp3 and Nsp3 (590–1,215 aa) was significantly higher than that of Nsp3 (1–418 aa) in both HEK-293 T and IPEC-J2 cells (Fig. 7a). The effect of Nsp3 (590–1,215 aa) transfection on IκBα ubiquitination was evaluated by transfecting the Nsp3 (590–1,215 aa) and pCMV-HA-Ub eukaryotic expression plasmids into the IPEC-J2 and HEK-293 T cells. The cell extracts were subjected to co-immunoprecipitation. As shown in Fig. 7b, Nsp3 (590–1,215 aa) transfection decreased the IκBα ubiquitination levels. These results indicate that Nsp3 can induce deubiquitination and that the amino acids at positions 590–1,215 in Nsp3 may inhibit the degradation of IκBα by decreasing the IκBα ubiquitination levels, which results in the suppression of NF-κB signaling pathway.