Porcine reproductive and respiratory syndrome virus inhibits type I interferon signaling by blocking STAT1/STAT2 nuclear translocation

Abstract

Type I interferons (IFNs) IFN-α/β play an important role in innate immunity against viral infections by inducing antiviral responses. Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the synthesis of type I IFNs. However, whether PRRSV can inhibit IFN signaling is less well understood. In the present study, we found that PRRSV interferes with the IFN signaling pathway. The transcript levels of IFN-stimulated genes ISG15 and ISG56 and protein level of signal transducer and activator of transcription 2 (STAT2) in PRRSV VR2385-infected MARC-145 cells were significantly lower than those in mock-infected cells after IFN-α treatment. IFN-induced phosphorylation of both STAT1 and STAT2 and their heterodimer formation in the PRRSV-infected cells were not affected. However, the majority of the STAT1/STAT2/IRF9 (IFN regulatory factor 9) heterotrimers remained in the cytoplasm of PRRSV-infected cells, which indicates that the nuclear translocation of the heterotrimers was blocked. Overexpression of NSP1β of PRRSV VR2385 inhibited expression of ISG15 and ISG56 and blocked nuclear translocation of STAT1, which suggests that NSP1β might be the viral protein responsible for the inhibition of IFN signaling. PRRSV infection in primary porcine pulmonary alveolar macrophages (PAMs) also inhibited IFN-α-stimulated expression of the ISGs and the STAT2 protein. In contrast, a licensed low-virulence vaccine strain, Ingelvac PRRS modified live virus (MLV), activated expression of IFN-inducible genes, including those of chemokines and antiviral proteins, in PAMs without the addition of external IFN and had no detectable effect on IFN signaling. These findings suggest that PRRSV interferes with the activation and signaling pathway of type I IFNs by blocking ISG factor 3 (ISGF3) nuclear translocation.

FIG. 1.

PRRSV inhibits expression of IFN-stimulated genes in MARC-145 cells. (A) Reduction of ISG15 and ISG56 transcripts, as detected by real-time RT-PCR. Cells were inoculated with PRRSV VR2385, incubated for 24 h, and then treated with IFN-α for 1 h. (B) UV-inactivated PRRSV has no effect on expression of ISG15 and ISG56, as detected by real-time RT-PCR. Cells were inoculated with PRRSV VR2385 or UV-inactivated VR2385, incubated for 24 h, and then treated with IFN-α for 1 h. (C) Reduction of ISG15 and ISG56 transcripts in PRRSV-infected cells 15 h after IFN treatment. Cells were inoculated with PRRSV VR2385, incubated for 24 h, and then treated with IFN-α for 15 h. Significant differences between the two groups for each transcript are denoted by a single asterisk and a double asterisk, which indicate P values of <0.05 and <0.01, respectively. Error bars represent standard deviations. (D) Inhibition of IFN-induced STAT2 protein expression by Western blotting. Cells were infected with PRRSV VR2385 or mock infected for 24 h and then treated with IFN-α for 8 h. The same blot was incubated with β-tubulin antibody as a protein loading control. (E) Western blotting of STAT1 and STAT2 from the cells 24 h after IFN-α treatment in the presence or absence of PRRSV infection.

FIG. 2.

Phosphorylation status of STAT1 and STAT2 in PRRSV-infected MARC-145 cells after IFN-α stimulation. (A) Western blotting with antibodies against STAT1-Y701 and STAT2-Y690. Cells were infected with PRRSV VR2385 or mock infected and, at 24 hpi, treated with IFN-α for 1 h. The same blot was incubated with β-tubulin antibody as a protein loading control. Convalescent pig antiserum was used to blot the membrane to show the PRRSV proteins in the lysates of PRRSV-infected cells. Positions of prestained molecular mass markers are shown on the left. (B) Western blotting with antibodies against STAT1-Y701 and STAT2-Y690 in VR2385-infected or mock-infected cells 0.5, 2, and 8 h after IFN-α treatment.

FIG. 3.

IP detection of STAT1/STAT2 heterodimer formation in MARC-145 cells after IFN-α treatment. Cells were infected with PRRSV VR2385 or mock infected and, at 24 hpi, treated with IFN-α for 1 h. WB, Western blot.

FIG. 4.

Blockage of nuclear translocation of ISGF3 heterotrimers in PRRSV-infected MARC-145 cells. (A) PRRSV inhibits nuclear translocation of STAT1-eGFP, as observed by confocal microscopy. Cells were transiently transfected with STAT1-eGFP plasmid and inoculated with VR2385 4 h later. Cells were treated with IFN-α at 24 hpi and fixed 1 h later. (B) Phosphorylated STAT1 and STAT2 in nuclear and cytoplasmic fractions. Subcellular fractionation of the cells 1 h after IFN treatment and Western blotting was determined. The same blot was incubated with antibodies against β-tubulin and histone H1 as controls for loading and fractionation. (C) Densitometry analysis of the digital image shown in panel B. The band intensity of each fraction is shown as the relative percentage of the sum density of corresponding cytoplasmic and nuclear fractions from the same treatment. Normalization for cytoplasmic and nuclear fractions was done with tubulin and histone H1, respectively.

FIG. 5.

PRRSV NSP1β protein inhibits IFN signaling. (A) NSP1β inhibits expression of ISG15 and ISG56 in HEK293 cells. Cells were transiently transfected with NSP1α and NSP1β plasmids or an empty vector and, 48 h after transfection, treated with IFN-α at 300 U/ml. The cells were harvested 12 h after IFN treatment. Significant differences in ISG15 and ISG56 transcript levels between the two groups of NSP1β and empty vector are denoted by an asterisk, which indicates a P value of <0.05. (B) NSP1α and NSP1β have no effect on IFN-induced phosphorylation of STAT1 in HEK293 cells. Cells were harvested for STAT1-Y701 detection 1 h after IFN treatment. (C) NSP1β inhibits nuclear translocation of STAT1-eGFP in HeLa cells, as observed by confocal microscopy. Cells were transiently transfected with STAT1-eGFP and NSP1β-RFP plasmids. At 24 h after transfection, the cells were treated with IFN-α at 300 U/ml for 1 h. (D) NSP1β inhibits STAT1 nuclear translocation. HEK293 cells were transiently transfected with NSP1β plasmid or an empty vector and, 48 h after transfection, treated with IFN-α at 300 U/ml for 1 h. Subcellular fractionation of the cells and Western blotting were conducted to detect phosphorylated STAT1 in nuclear and cytoplasmic fractions. The same blot was incubated with antibodies against NSP1β, β-tubulin, and histone H1 as controls for loading and fractionation. EV, empty vector; 1α, NSP1α; 1β, NSP1β.

FIG. 6.

VR2385 interferes with IFN signaling in PAMs. (A) Real-time RT-PCR detection of ISG15 and IFI56 from PAMs 8 h after IFN-α treatment in the presence or absence of VR2385 infection. Significant differences between the two IFN-treated groups are denoted by an asterisk, which indicates a P value of <0.05. (B) Western blotting of STAT2 from PAMs 8 h after IFN-α treatment in the presence or absence of VR2385 infection. (C) Western blotting with the antibodies against phosphorylated STAT1 and STAT2 from PAMs 1 h after IFN-α treatment in the presence or absence of VR2385 infection.

FIG. 7.

Effect of PRRSV MLV on IFN signaling in PAMs. (A) Real-time RT-PCR detection of ISG15 and IFI56 from PAMs 8 h after IFN-α treatment in the presence or absence of PRRSV infection. Significant differences in the transcript levels between the IFN-treated groups and the nontreated groups are denoted by a single asterisk and a double asterisk, which indicate P values of <0.05 and <0.01, respectively. (B) Western blotting of STAT2 in PAMs 8 h after IFN-α treatment in the presence or absence of PRRSV infection. Samples of mock-treated PAMs were included as controls. VR, VR2385. (C) Real-time RT-PCR detection of CXCL10 transcript in PAMs 8 h after IFN-α treatment in the presence or absence of PRRSV infection. Significant differences between VR2385-infected and mock-infected cells are denoted by an asterisk, which indicates a P value of <0.05. (D) Real-time RT-PCR detection of CCL2, MX1, OAS2, and RNase L transcripts in PAMs 8 h after IFN-α treatment in the presence or absence of PRRSV infection. Significant differences between virus-infected and mock-infected cells are denoted by a single asterisk and a double asterisk, which indicate P values of <0.05 and <0.01, respectively. (E) Real-time RT-PCR detection of ISG56 from HEK293 cells transfected with MLV NSP1α and NSP1β plasmids or empty vector pCMVTag2B. At 48 h after the transfection, the cells were treated with IFN-α at 300 U/ml for 12 h. No significant difference in ISG56 transcript level between the samples was detected.