Figure 1.
PCV2 Rep enhances the production of IL-10 in PAMs. (A) Model of construction of PCV1-Rep2 and PCV2-Rep1. The gray and dark lines represent the PCV1 and PCV2 backbones, respectively. (B) PAMs were infected with Mock, PCV1, PCV2, PCV2-Rep1, or PCV1-Rep2, and IL-10 production was analyzed by ELISA for 0–24 h, 24–48 h, and 48–72 h p.i. The columns indicate IL-10 production in each 24 h in the culture supernatants. (C) The IL-10 mRNA levels of the PAMs infected with Mock, PCV1, PCV2, PCV2-Rep1, or PCV1-Rep2 were detected by quantitative PCR at the indicated times post-infection. The results are mean ± SEM of three independent experiments. **P < 0.01 versus Mock infection at the same infection time point. #p < 0.05, ##p < 0.01 versus PCV2-infected cells at the same infection time point. &&p < 0.01 versus PCV1 infection at the same infection time point.
Figure 1.
PCV2 Rep enhances the production of IL-10 in PAMs. (A) Model of construction of PCV1-Rep2 and PCV2-Rep1. The gray and dark lines represent the PCV1 and PCV2 backbones, respectively. (B) PAMs were infected with Mock, PCV1, PCV2, PCV2-Rep1, or PCV1-Rep2, and IL-10 production was analyzed by ELISA for 0–24 h, 24–48 h, and 48–72 h p.i. The columns indicate IL-10 production in each 24 h in the culture supernatants. (C) The IL-10 mRNA levels of the PAMs infected with Mock, PCV1, PCV2, PCV2-Rep1, or PCV1-Rep2 were detected by quantitative PCR at the indicated times post-infection. The results are mean ± SEM of three independent experiments. **P < 0.01 versus Mock infection at the same infection time point. #p < 0.05, ##p < 0.01 versus PCV2-infected cells at the same infection time point. &&p < 0.01 versus PCV1 infection at the same infection time point.
Figure 2.
PCV2 Rep rather than PCV1 Rep directly promotes IL-10 production in PAMs. (A) 1 × 106 of PAMs were infected with 100 MOI recombinant adenoviruses rAd-Blank, rAd-Rep1, rAd-Rep2, or Mock infection, and then the expressions of Rep were measured by western blotting. (B,C) IL-10 secretion and mRNA levels were detected by ELISA and Q-PCR, respectively. (D) The activities of il10 promoter were measured in the PAMs co-transfected with the Rep1 protein expression plasmid, Rep2 protein expression plasmid, or blank vector (control) by the dual-luciferase reporter assay system. The data of (A) are representative of three independent experiments. The data of (B–D) are the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 versus Mock infection at same infection time point (B) or control (D). ##p < 0.01 versus rAd-Blank-infected cells at the same infection time point (B,C) or Rep1 (D). &p < 0.05, &&p < 0.01 versus rAd-Rep1-infected cells at the same infection time point.
Figure 2.
PCV2 Rep rather than PCV1 Rep directly promotes IL-10 production in PAMs. (A) 1 × 106 of PAMs were infected with 100 MOI recombinant adenoviruses rAd-Blank, rAd-Rep1, rAd-Rep2, or Mock infection, and then the expressions of Rep were measured by western blotting. (B,C) IL-10 secretion and mRNA levels were detected by ELISA and Q-PCR, respectively. (D) The activities of il10 promoter were measured in the PAMs co-transfected with the Rep1 protein expression plasmid, Rep2 protein expression plasmid, or blank vector (control) by the dual-luciferase reporter assay system. The data of (A) are representative of three independent experiments. The data of (B–D) are the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 versus Mock infection at same infection time point (B) or control (D). ##p < 0.01 versus rAd-Blank-infected cells at the same infection time point (B,C) or Rep1 (D). &p < 0.05, &&p < 0.01 versus rAd-Rep1-infected cells at the same infection time point.
Figure 3.
PCV2 Rep specifically enhances the activity of the p38-MAPK signaling pathway to promote IL-10 expression in PAMs. (A) PAMs were respectively infected with 100 MOI rAd-Blank (Blank) or rAd-Rep2 (Rep2) for 0 h, 24 h, and 48 h, and the expression and the phosphorylation levels of Akt, ERK, and p38-MAPK were detected by western blotting. (B) PAMs transfected with the specific siRNAs of Akt, ERK, p38-MAPK, or NC were infected with Mock, rAd-Blank, or rAd-Rep2 for 48 h, and the production of IL-10 was measured by the ELISA assay. (C) PAMs were transfected with the same siRNAs as B and then inoculated with 5 MOI PCV2 for 24 h. The production of IL-10 was detected by the ELISA assay. The data of (A) are representative of three independent experiments. The data of (B,C) are the means ± SEM of three independent experiments. **p < 0.01 versus NC siRNA-transfected cells.
Figure 3.
PCV2 Rep specifically enhances the activity of the p38-MAPK signaling pathway to promote IL-10 expression in PAMs. (A) PAMs were respectively infected with 100 MOI rAd-Blank (Blank) or rAd-Rep2 (Rep2) for 0 h, 24 h, and 48 h, and the expression and the phosphorylation levels of Akt, ERK, and p38-MAPK were detected by western blotting. (B) PAMs transfected with the specific siRNAs of Akt, ERK, p38-MAPK, or NC were infected with Mock, rAd-Blank, or rAd-Rep2 for 48 h, and the production of IL-10 was measured by the ELISA assay. (C) PAMs were transfected with the same siRNAs as B and then inoculated with 5 MOI PCV2 for 24 h. The production of IL-10 was detected by the ELISA assay. The data of (A) are representative of three independent experiments. The data of (B,C) are the means ± SEM of three independent experiments. **p < 0.01 versus NC siRNA-transfected cells.
Figure 4.
PCV2 Rep activates p38-MAPK signaling to promote NF-κB p50 and Sp1 binding to the il10 promoter. (A–C) PAMs were infected with 100 MOI rAd-Rep1 (Rep1), rAd-Rep2 (Rep2), rAd-Blank (Blank), or Mock infection, and the binding levels of transcriptional factor NF-κB p50, Sp1, and AP1 to the il10 promoter were detected by the ChIP assay. (D,E) PAMs were transfected with the specific siRNAs of p50, p38-MAPK, or NC, and then infected with rAd-Rep2. The binding activities of p50 and Sp1 to the il10 promoter were detected by the ChIP assay. The data are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus rAd-Blank-infected cells (A,B) or NC siRNA-transfected cells in the same rAd group (D,E). ##p < 0.01 versus rAd-Rep1-infected cells.
Figure 4.
PCV2 Rep activates p38-MAPK signaling to promote NF-κB p50 and Sp1 binding to the il10 promoter. (A–C) PAMs were infected with 100 MOI rAd-Rep1 (Rep1), rAd-Rep2 (Rep2), rAd-Blank (Blank), or Mock infection, and the binding levels of transcriptional factor NF-κB p50, Sp1, and AP1 to the il10 promoter were detected by the ChIP assay. (D,E) PAMs were transfected with the specific siRNAs of p50, p38-MAPK, or NC, and then infected with rAd-Rep2. The binding activities of p50 and Sp1 to the il10 promoter were detected by the ChIP assay. The data are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus rAd-Blank-infected cells (A,B) or NC siRNA-transfected cells in the same rAd group (D,E). ##p < 0.01 versus rAd-Rep1-infected cells.
Figure 5.
The Rep protein activates p38-MAPK at the later phase of PCV2 infection. (A–D) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the p-p38-MAPK were detected by western blotting at 12 h, 24 h, and 48 h of post-inoculation (A); the relevant statistical results of p-p38-MAPK, Rep1, and Rep2 expression are shown in histograms (B–D). The data of (A) are representative of three independent experiments. The data of (B–D) are the means ± SD of three independent experiments. **p < 0.01 versus PCV2-inoculated cells. &&p < 0.01 versus PCV2-Rep1-inoculated cells. ##p < 0.01 versus PCV1-inoculated cells.
Figure 5.
The Rep protein activates p38-MAPK at the later phase of PCV2 infection. (A–D) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the p-p38-MAPK were detected by western blotting at 12 h, 24 h, and 48 h of post-inoculation (A); the relevant statistical results of p-p38-MAPK, Rep1, and Rep2 expression are shown in histograms (B–D). The data of (A) are representative of three independent experiments. The data of (B–D) are the means ± SD of three independent experiments. **p < 0.01 versus PCV2-inoculated cells. &&p < 0.01 versus PCV2-Rep1-inoculated cells. ##p < 0.01 versus PCV1-inoculated cells.
Figure 6.
Rep protein enhances the binding activities of p50 and Sp1 with the il10 promoter at the later phase of PCV2 infection. (A–D) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the binding activities of p50 and Sp1 were detected at 12 h, 24 h, and 48 h using the ChIP assay (A,B); the relevant statistical results of (A,B) are shown in line graphs (C,D). The data of (A,B) are representative of three independent experiments. The data of (C,D) are the means ± SD of three independent experiments. **p < 0.01 represents the PCV2-inoculated cells versus the PCV1-Rep2-inoculated cells at the same infection time point. #p < 0.05 represents the PCV2-inoculated cells versus the PCV2-Rep1-inoculated cells at the same infection time point. &p < 0.05 versus PCV1-inoculated cells at the same infection time point.
Figure 6.
Rep protein enhances the binding activities of p50 and Sp1 with the il10 promoter at the later phase of PCV2 infection. (A–D) PAMs were infected with 5 MOI PCV1, PCV2, PCV2-Rep1, and PCV1-Rep2, and the binding activities of p50 and Sp1 were detected at 12 h, 24 h, and 48 h using the ChIP assay (A,B); the relevant statistical results of (A,B) are shown in line graphs (C,D). The data of (A,B) are representative of three independent experiments. The data of (C,D) are the means ± SD of three independent experiments. **p < 0.01 represents the PCV2-inoculated cells versus the PCV1-Rep2-inoculated cells at the same infection time point. #p < 0.05 represents the PCV2-inoculated cells versus the PCV2-Rep1-inoculated cells at the same infection time point. &p < 0.05 versus PCV1-inoculated cells at the same infection time point.
Figure 7.
Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. (A) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. (B) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 106 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. (C) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. (D) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. (E,F) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of (A) are representative of three independent experiments. The data of (B–D) are means ± SEM of three independent experiments. The data of (E,F) are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus NC siRNA-transfected cells in the same infection (B,E,F) or at the same time point (C,D).
Figure 7.
Rep protein interacts with thymine DNA glycosylase (TDG) to enhance the binding activities of Sp1 and NF-κB p50 with the il10 promoter at the later phase of PCV2 infection. (A) These specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q were transfected to cells for 48 h, and the efficiency of each gene silencing was detected by western blotting. (B) The specific siRNAs of c-Myc, ZNF265, TDG, and VG5Q siRNAs (siRNA #1 of c-Myc, siRNA #2 of ZNF265, siRNA #3 of TDG, and siRNA #2 of VG5Q) were transfected to PAMs, then rAd-Blank or rAd-Rep2 infected the cells (1 × 106 cells) for 48 h. The secretion of IL-10 was measured by ELISA in different siRNA-transfected-PAMs. (C) The secretion of IL-10 was detected in the TDG siRNA-transfected-PAMs in 0–24 h, 24–48 h, and 48–72 h after PCV2 inoculation by ELISA. The columns indicate IL-10 production in each 24 h in the culture supernatants. (D) The levels of IL-10 mRNA were detected in TDG siRNA-transfected-PAMs by Q-PCR with the same incubation time points as C. (E,F) TDG siRNA-transfected-PAMs were incubated with 5 MOI PCV2, the binding activities of NF-κB p50 and Sp1 to the il10 promoter were detected at 48 h by the ChIP assay. The data of (A) are representative of three independent experiments. The data of (B–D) are means ± SEM of three independent experiments. The data of (E,F) are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus NC siRNA-transfected cells in the same infection (B,E,F) or at the same time point (C,D).

Table 1.
siRNA sequences for target genes used in this study.
Table 1.
siRNA sequences for target genes used in this study.
Target Gene | Accession Number | Sequences |
---|
Akt | NM_001159776.1 | AACGAGGCGAGUACAUCAAGATT |
p38 | XM_001929490.5 | AAGCUAUCCAGACCAUUUCAATT |
ERK | NM_001198922.1 | AAGCACCAUUCAAGUUUGACATT |
p50 | KC316024.1 | AAGGAGGAGAAUUACAGGUUCTT |
VG5Q | XM_003123715.5 | 1# GCAAGACCCAUACAAGCAATT |
| | 2# CCGUAUUUGUUCCAUGUAATT |
| | 3# GCAGGUAACUGCCAGAUAATT |
TDG | XM_021092634.1 | 1# GCAUAAACCUAGAUGCACUTT |
| | 2# GGUAGAAGCGUAGUGGCCUTT |
| | 3# GCCAGAGACUAUAGAAGACTT |
ZNF265 | NM_001044582.1 | 1# CCAGAAGAUCAGAGUGUAATT |
| | 2# CCUAUAUUAAGGGUGCCUUTT |
| | 3# GCCUAACGGUUCAUCUCUUTT |
c-Myc | NM_001005154.1 | 1# CCAUGAAUUCACACUUGUUTT |
| | 2# GCAUGAUCCAGUGCAACCUTT |
| | 3# GCAAACUUUCCUCUGUAAATT |