Figure 1.
PRRSV VR-2385 induces elevation of phosphorylated STAT1 at serine 727 (pSTAT1-S727) in MARC-145 cells.
A. VR-2385 (VR) induces higher level of pSTAT1-S727 in MARC-145 cells than MLV. The infected cells were harvested at 24 hpi for Western blotting with antibodies against pSTAT1-S727, STAT1 and tubulin. Lysate of mock-infected cells was included as a control. B. Densitometry analysis of pSTAT1-S727 bands to show relative level of pSTAT1 in comparison with mock-infected cells as folds after normalization with tubulin. Significant difference from mock-infected control is shown by “**”, which indicates P<0.01. Errors bars indicate standard errors of repeated experiments. C. PRRSV viral RNA copies in the PRRSV-infected cells detected by real-time RT-qPCR and shown as log10 per 10 ng total RNA.
Figure 2.
Kinetics and dose-dependent effect of PRRSV-induced elevation of pSTAT1-S727 (pSTAT1).
A. Kinetics of pSTAT1-S727 in VR-2385-infected cells. MARC-145 cells were infected with VR-2385 and MLV. The cells were harvested at 12, 24 and 48 hpi for Western blotting. Relative levels of pSTAT1 in comparison to the mock-infected cells of 12 hpi time point are shown as folds below the images. B. The pSTAT1 level increases along with incremental addition of VR-2385 virus inoculum. The MARC-145 cells were infected with 0.01, 0.1, 0.5, 1.0 and 2.0 MOI and harvested at 24 hpi for Western blotting. C. Densitometry analysis of pSTAT1 showing relative levels of pSTAT1 in comparison with cells inoculated with 0.01 MOI as folds after normalization with tubulin. Error bars indicate standard errors among three repeated experiments.
Figure 3.
The p38 MARK signaling pathway is involved in pSTAT1-S727 elevation in PRRSV-infected cells.
A. SB203580 treatment leads to inhibition of PRRSV-induced pSTAT1 elevation. MARC-145 cells were infected with PRRSV and treated with SB203580. At 24 hpi, the cells were harvested for Western blotting with antibodies against pSTAT1-S727, STAT1, and tubulin. B. PRRSV yield remains unchanged in the presence of SB203580. Cell culture supernatant samples of PRRSV-infected MARC-145 cells in the presence or absence of SB203580 were titrated. The viral yield is shown as log10 TCID50/ml. C. Cell viability assay of SB203580-treated MARC-145 cells. The cells were subjected for the assay 24 h after treatment. Relative percentages in comparison with mock-treated cells are shown.
Figure 4.
VR-2385 infection leads to higher expression of proinflammatory cytokine genes than MLV and SB203580 treatment reduces their expression.
MARC-145 cells were infected with VR-2385 or MLV and treated with SB203580. Mock-treated cells were included for control. The cells were harvested at 48 hpi for RNA isolation and RT-qPCR. Relative folds of transcript levels of IL-1β, IL-8, and ISG54 in comparison with mock-treated PRRSV-negative cells are shown. Error bars represent variation of three repeated experiments. Significant differences between paired samples are shown by “*” and “**”, which indicate P<0.05 and P<0.01, respectively.
Figure 5.
Inhibition of PRRSV-mediated STAT1 phosphorylation by methylthioadenosine (MTA).
A. MTA treatment leads to blockage of VR-2385-induced STAT1 phosphorylation. MARC-145 cells were infected with VR-2385 and treated with MTA. The cells were harvested at 24 hpi for Western blotting with antibodies against pSTAT1-S727, STAT1, and tubulin. Relative levels of pSTAT1 in comparison to the mock-treated and uninfected cells are shown as folds below the images. B. PRRSV yield remains stable in the presence of MTA. The virus samples were titrated and the result is shown as log10 TCID50/ml. C. MTA treatment reduces PRRSV-induced expression of proinflammatory cytokine genes. MARC-145 cells were infected with VR-2385 and treated with MTA. The cells were harvested at 48 hpi for RNA isolation and RT-qPCR. “*” indicates significant differences (P<0.05) between MTA-treated and mock-treated cells. D. Cell viability assay of MTA-treated MARC-145 cells. The cells were subjected for the assay 24 h after treatment. Relative percentages in comparison with mock-treated cells are shown.
Figure 6.
PRRSV infection of PAM cells leads to elevation of pSTAT1-S727.
A. Elevation of pSTAT1-S727 in VR-2385-infected PAM cells detected by Western blotting. The cells were infected with VR-2385 and treated with SB203580. Mock-infected and mock-treated cells were included as controls. The blotting was done with antibodies against pSTAT1-S727, STAT1 and tubulin. Relative levels of pSTAT1-S727 compared to the mock-treated control are shown as folds below the images. B. PRRSV viral RNA copies in PAMs detected by RT-qPCR and shown as log10 per 10 ng total RNA. C&D. Increased expression of IL-1β, CXCL10, IL-8, IL-10, and CCL2 in PRRSV-infected PAMs detected by RT-qPCR. The cells were infected with VR-2385 and treated with SB203580. Relative levels of gene expression are shown as folds in comparison with the mock-treated PRRSV-negative cells.
Figure 7.
Screening of non-structural and structural proteins of PRRSV VR-2385 in inducing pSTAT1-S727 elevation.
A. Screening of VR-2385 non-structural proteins (nsps) 1–12. HEK293 cells were transfected with STAT1-GFP and VR-2385 nsp plasmids. The cells were harvested 48 h after transfection for Western blotting with antibodies against pSTAT1-S727, STAT1 and tubulin. Relative levels of pSTAT1 in comparison with control vector lane are shown as folds below the images. B. Screening of VR-2385 structural proteins (sps) encoded by ORFs 2–7. HEK293 cells were transfected with STAT1-GFP and VR-2385 sp plasmids. Blotting and analysis were conducted similarly as in “A”. C. Expression of endogenous STAT1 remains stable in HEK293 cells transfected with PRRSV nsps. The cells were transfected with empty vector, GFP, nsp1α, nsp5, nsp8 or nsp11 plasmids. Western blotting with antibodies against STAT1 and tubulin was conducted.
Figure 8.
Nsp12 induces elevation of pSTAT1-S727 and expression of proinflammatory genes in HEK293 cells.
A. Nsp12 induces elevation of pSTAT1-S727 in HEK293 cells. The cells were transfected with nsp12 or nps4 plasmids. The cells were harvested 48 h after transfection for Western blotting with antibodies against pSTAT1-S727, STAT1 and tubulin. B. Densitometry analysis showing relative pSTAT1 levels in comparison with empty vector lane after normalization with tubulin. C. Nsp12 induces expression of proinflammatory cytokine genes. HEK293 cells were transfected with nsp12 plasmid and harvested for RT-qPCR. Significant differences between cells transfected with nsp12 and those with empty vector are shown by “*”, which indicates P<0.05.
Table 1.
List of primers for real-time PCR.