Expression Profile of Porcine TRIM26 and Its Inhibitory Effect on Interferon-β Production and Antiviral Response

Abstract

TRIM26, a member of the tripartite motif (TRIM) family has been shown to be involved in modulation of innate antiviral response. However, the functional characteristics of porcine TRIM26 (porTRIM26) are unclear. In this study, we used a synthesized antigen peptide to generate a polyclonal antibody against porTRIM26 with which to study the expression and function of porTRIM26. We demonstrated that polyinosinic:polycytidylic acid (poly (I:C)) stimulation and viral infection (vesicular stomatitis (VSV) or porcine reproductive and respiratory syndrome virus (PRRSV)) induce expression of porTRIM26, whereas knock-down expression of porTRIM26 promotes interferon (IFN)- production after poly (I:C) stimulation and virus infection (VSV or PRRSV). The importance of the porTRIM26-mediated modulation of the antiviral response was also shown in VSV- or PRRSV-infected cells. In summary, these findings show that porTRIM26 has an inhibitory role in IFN- expression and the antiviral response.

Keywords: IFN-; PRRSV; TRIM26; VSV; antiviral response; poly (I:C).

Figure 1

Multiple alignment and phylogenetic analysis of TRIM26. (A) Polypeptide of porcine TRIM26 was aligned with that of human, mouse, and rat. The RING finger domain (RING), the B-box domains (B-box), and the coiled-coil region (CC) are labeled in reference to the human TRIM26. The antigenic peptide is shown in the red box. (B) The phylogenetic tree of TRIM26, constructed by neighbor-joining method using MEGA 6.06.

Figure 2

(A) Specificity of polyclonal antibody against porcine TRIM26 was analyzed with Western blotting. (B) Expression profiles of TRIM26 in different porcine cell lines, analyzed with Western blotting with anti-TRIM26 antibody. (C) Expression profiles of TRIM26 in different pig tissues (n = 3), analyzed with Western blotting and an anti-TRIM26 polyclonal antibody. Approximately 100 mg of each tissue was homogenized with lysis buffer as per the Materials and Method section for protein sample preparation. (D) Expression profiles of TRIM26 in different pig tissues (n = 3) analyzed by reverse transcription-quantitative PCR.

Figure 3

Effect of porTRIM26 on poly (I:C)-induced interferon (IFN)-β. (A) Expression of TRIM26 in porcine alveolar macrophages (PAM) or porcine iliac artery endothelial cells (PIEC) with or without poly(I:C) treatment was measured with qPCR and Western blotting. Poly (I:C) treatment induced the expression of TRIM26 in both PAM and PIEC cells. (B) PAM were transfected with porTRIM26 siRNA or negative control (NC) siRNA for 72 h and then stimulated with poly (I:C) for 6 h. Supernatant and cells were harvested and analyzed by enzyme-linked immunosorbent assay (ELISA) and qPCR, respectively. (C) PIEC cells were transfected with porTRIM26 siRNA or NC siRNA for 72 h, and then stimulated with poly (I:C) for 6 h. Supernatant and cells were harvested and analyzed by ELISA and qPCR, respectively. (D) PIEC cells were transfected with plasmid encoding porTRIM26 or the empty vector. After 24 h, the cells were treated with poly (I·C) or vehicle and incubated for another 6 h. Level of IFN-β mRNA was determined with RT-qPCR. Data present are mean ± SEM pooled from one independent experiment; n ≥ 3 for each of the analyzed parameters. *, p < 0.05; **, p < 0.01; ***, p < 0.001 in comparison between mock and poly (I:C) group (A); between NC and RNAi with poly (I:C) stimulation (B,C); and between the empty vector and pFlag-porTRIM26 with poly (I:C) stimulation (D).

Figure 4

Effect of porTRIM26 on IFN-β expression and vesicular stomatitis virus (VSV) infection in PIEC cells. (A) PIEC cells were infected with VSV at a multiplicity of infection (MOI) of 1 for 24 h. The porTRIM26 mRNA and protein level was measured by RT-qPCR and Western blotting, respectively. (B) PIEC cells were transfected with porTRIM26 siRNA or NC siRNA. After 72 h, the cells were mock infected or infected with VSV at a MOI of 1. Supernatants were collected at 24 h post infection (hpi) for tissue culture infective dose (TCID₅₀) assay. (C) PIEC cells were transfected with plasmid encoding porTRIM26 or empty vector for 24 h and then infected with VSV at an MOI of 1. Supernatants were collected at 24 hpi for either a TCID₅₀ assay or ELISA. Data are mean ± SEM pooled from one independent experiment; n ≥ 3 for each of the analyzed parameters. ND, no detected. **, p < 0.01; ***, p < 0.001 in comparison: mock-infected vs. VSV-infected group (A); NC-treated vs. siRNA-treated groups after VSV infection (B); empty-vector-transfected vs. pFlag-pTRIM26-transfected after VSV infection (C).

Figure 5

Effect of porTRIM26 on IFN-β expression and porcine reproductive and respiratory syndrome virus (PRRSV) infection in PAM. (A) PAM were infected with PRRSV at a MOI of 1 for 24 h. The mRNA levels of TRIM26 and PRRSV ORF7 were measured with RT-qPCR. The protein levels of TRIM26 and PRRSV N were measured with Western blotting. PRRSV infection induced expression of TRIM26. (B) PAM were transfected with the porTRIM26 siRNA or NC siRNA. After 72 h, cells were mock-infected or infected with PRRSV at an MOI of 1. Supernatants were collected at 24 hpi for either a TCID₅₀ assay or ELISA. The knock-down of porTRIM26 expression increased the production of IFN-b and inhibited PRRSV infection. Data are mean ± SEM pooled from one independent experiment; n ≥ 3 for each of the analyzed parameters. ND: not detected. **, p < 0.01; ***, p < 0.001 in comparison: mock-infected vs. PRRSV-infected group (A); NC-siRNA-transfected vs. porTRIM26-siRNA-transfected cells after infection with PRRSV (B).