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. 2016 Jan 12:6:19226.
doi: 10.1038/srep19226.

Exogenous avian leukosis virus-induced activation of the ERK/AP1 pathway is required for virus replication and correlates with virus-induced tumorigenesis

Manman Dai  1 , Min Feng  2 , Yu Ye  1 , Xiaochan Wu  1 , Di Liu  1 , Ming Liao  1   3   4 , Weisheng Cao  1   3   4
Affiliations

Exogenous avian leukosis virus-induced activation of the ERK/AP1 pathway is required for virus replication and correlates with virus-induced tumorigenesis

Manman Dai et al. Sci Rep. .

Abstract

A proteomics approach was used to reveal the up-regulated proteins involved in the targeted mitogen-activated protein kinase (MAPK) signal transduction pathway in DF-1 cells after ALV subgroup J (ALV-J) infection. Next, we found that ALV-J CHN06 strain infection of DF-1 cells correlated with extracellular signal-regulated kinase 2 (ERK2) activation, which was mainly induced within 15 min, a very early stage of infection, and at a late infection stage, from 108 h to 132 h post-infection. Infection with other ALV subgroup (A/B) strains also triggered ERK/MAPK activation. Moreover, when activating ERK2, ALV subgroups A, B and J simultaneously induced the phosphorylation of c-Jun, an AP1 family member and p38 activation but had no obvious effect on JNK activation at either 15 min or 120 h. Interestingly, only PD98059 inhibited the ALV-induced c-Jun phosphorylation while SP600125 or SB203580 had no influence on c-Jun activation. Furthermore, the viral gp85 and gag proteins were found to contribute to ERK2/AP1 activation. Additionally, the specific ERK inhibitor, PD980509, significantly suppressed ALV replication, as evidenced by extremely low levels of ALV promoter activity and ALV-J protein expression. In vivo analysis of ERK2 activation in tumor cells derived from ALV-J-infected chicken demonstrated a strong correlation between ERK/MAPK activation and virus-associated tumorigenesis.

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Figures

Figure 1
Figure 1. MAPK signaling pathway analysis of proteins that were significantly altered in ALV-J-infected DF-1 cells.
Red means up-regulated proteins. The mapped pathway of the increased proteins in ALV-J-infected DF-1 cells was obtained from the KEGG PATHWAY Database (http://www.genome.jp/kegg-bin/show_pathway?map04010).
Figure 2
Figure 2. Confirmation of MAPK protein levels by western blotting.
DF-1 cells were infected by live viral stocks of ALV-J CHN06 for 60 h at a multiplicity of infection (MOI) of 1. Mock-infected DF-1 cells were used as a negative control. Cell lysates were subjected to western blotting with rabbit anti-p44/42 MAPK and GAPDH antibodies. iTRAQ ratios are shown on the right side.
Figure 3
Figure 3. ALV infection activates ERK2 phosphorylation.
DF-1 cells were infected by live viral stocks of ALV-J CHN06 (104 TCID50 0.2 ml−1), ALV-J NX0101 (104 TCID50 0.2 ml−1), ALV-A GD13-1 (104 TCID50 0.2 ml−1), ALV-B CD08 (104 TCID50 0.2 ml−1) or wild strains of ALV-J KNE131218 (S/P ratio of 1.8) and JX130911A7 (S/P ratio of 2.2). Mock-infected DF-1 cells in various phases were used as a negative control. Cell lysates prepared at the indicated times p.i. were subjected to SDS-PAGE and the amounts of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 were evaluated by western blotting. Chickens solely express the 42-kDa ERK2 isoform. (a) The activation profile of ERK2 was explored in CHN06-infected DF-1 cells. (b) Viral stocks of CHN06 were filtered and pelleted. The filtrate, pellet and supernatant were used individually to infect DF-1 cells for 15 min and 120 h. (c) ERK2 phosphorylation was examined in DF-1 cells infected by ALV-A or B, NX0101 or wild strains of ALV-J, or various virus titers of CHN06, at 15 min or at 120 h p.i. The results are representative of two to three independent experiments.
Figure 4
Figure 4. ALV infection significantly activates AP-1 and p-p38, but not JNK phosphorylation, at 15 min and 120 h p.i.
DF-1 cells were mock infected or infected by CHN06, GD13-1 or CD08 at 104 TCID50 0.2 ml−1. (a) After 15 min or 120 h, cell lysates were collected and the amounts of p-c-Jun, c-Jun, p-JNK, total JNK, p-p38, total p38 and GAPDH were evaluated by western blotting. (b) DF-1 cells were treated with the JNK activator, Anisomycin (20 μM) for 12 hours according to the instructions, and cell lysates were collected and the amounts of p-JNK, total JNK were evaluated by western blotting. The results are representative of two to three independent experiments. Abbreviations: ANS, Anisomycin.
Figure 5
Figure 5. Drug cell toxicity test.
DF-1 cells were treated with LY294002 (10–20 μM), SB203580 (20 μM), SP600125 (10–20 μM), PD98059 (20–50 μM), U0126 (20 μM) or DMSO (0.1%, v/v). Cytotoxicity test was performed with Cell Counting Kit-8 dye. Data are representative of two independent experiments, both performed in triplicate. ***p < 0.001. Error bars indicate SEM. Abbreviations: LY, LY294002; SB, SB203580; SP, SP600125; PD, PD98059.
Figure 6
Figure 6. AP-1 is activated by ERK/MAPK pathway.
DF-1 cells were preincubated with 20 μM SP600125 (a), 20 μM SB203580 (b), 20 μM PD98059 (c) and 50 μM PD98059(d) for 1 h and subsequently infected with CHN06, GD13-1 or CD08 at 104 TCID50 0.2 ml−1. The inhibitors were in the cell culture fluid all the time. Cell lysates were collected and the amounts of p-c-Jun, c-Jun, VEGF-A and GAPDH were evaluated by western blotting. The results are representative of two independent experiments.
Figure 7
Figure 7. ALV infection activates ERK2 phosphorylation in a MEK-dependent manner.
DF-1 cells were preincubated with PD98059 (2.5–50 μM), U0126 (5–10 μM) or DMSO (0.1%, v/v) for 1 h and subsequently infected with CHN06 (a,b), GD13-1 (d) or CD08 (e) for 15 min, or with NX0101 (c) for 120 h, at 104 TCID50 0.2 ml−1. The inhibitors or DMSO were in the cell culture fluid all the time. And the concentration of inhibitor control is the maximum concentration used in each independent experiment. Cell lysates were collected and the amounts of p-ERK2 and total ERK were evaluated by western blotting. The results are representative of two independent experiments. Abbreviations: CHN, CHN06 or HN06; NX, NX0101; GD13-1, GD; CD08, CD.
Figure 8
Figure 8. gp85 or gag protein alone induces ERK2/AP1activation.
Cell lysates were analyzed by western blot analysis of kinase activation using antibodies specific for the phosphorylated forms of the proteins. (a) DF-1 cells were stimulated for 10 min with different concentrations of purified gp85, followed by western blot analysis of phosphorylated proteins and total proteins. (b) DF-1 cells were electroporated with pCMV-gag-EGFP or vector. Seventy-two hours later, cell lysates were subjected to western blotting with phosphorylated proteins, total proteinsp27 or GAPDH antibodies. (c) DF-1 cells were incubated for 1 h in the presence or absence of the PI3K inhibitor, LY294002 (20 μM), the ERK/MAPK inhibitor, PD98059 (20 μM), or DMSO (0.1%, v/v), and subsequently infected with CHN06 for 15 min at 104 TCID50 0.2 ml−1, followed by western blot analysis of ERK and Akt activation. Data shown are representative of two independent experiments
Figure 9
Figure 9. MAPK pathway regulates ALV envelope mRNA and cellular VEGF mRNA expression.
(a) DF-1 cells were treated with PD98059, U0126, SP600125, SB203580 (20 μM) or DMSO (0.1%, v/v) at the indicated times during the infection with CHN06. At 72 h p.i., viral production was monitored by real-time RT-PCR with primers designed for the envelope gene. (b) DF-1 cells were simultaneously incubated with CHN06 (104 TCID50 0.2 ml−1) and increasing concentrations of U0126 or DMSO (0.1%, v/v). Viral envelope gene and cellular VEGF transcription levels were measured by real-time RT-PCR at 72 h p.i. using special primers. (c) DF-1 cells were treated with SP600125, SB203580 (20 μM) or DMSO (0.1%, v/v) at the indicated times during the infection with CHN06. At 72 h p.i., the cellular VEGF transcription level was monitored by real-time RT-PCR with special primers. Data are representative of two independent experiments, both performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant. Error bars indicate SEM.
Figure 10
Figure 10. Effects of MAPK pathway inhibitors on ALV LTR promoter activity.
DF-1 cells were seeded in 24-well dishes and co-transfected with 0.2 μg pGL3-LTR and 0.02 μg pRL-TK. At 6 h p.i., cells were treated with PD98059 (red bar, 200 nM–20 μM), SP600125 (green bar, 200 nM–20 μM), SB203580 (purple bar, 5 μM–10 μM) or DMSO (second black bar, 0.1%, v/v). At 48 h p.i., cells were lysed for luciferase detection. Data are representative of three independent experiments, performed in triplicate. *p < 0.05, ***p < 0.001. Error bars indicate SEM.
Figure 11
Figure 11. Effects of MAPK pathway inhibitors on ALV-J p27 protein expression.
(a) DF-1 cells were simultaneously incubated with CHN06 (104 TCID50 0.2 ml−1) and PD98059, U0126 (20 μM) or DMSO (0.1%, v/v). (b) DF-1 cells were simultaneously incubated with CHN06 (104.5 TCID50 0.2 ml−1) and SP600125, SB203580 (20 μM) or DMSO (0.1%, v/v). The inhibitors or DMSO were in the cell culture fluid all the time. ALV-J production was measured by ELISA with anti-p27 antibodies. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant. Error bars indicate SEM.
Figure 12
Figure 12. Effects of the ERK pathway inhibitor on ALV-J gp85 protein expression.
DF-1 cells were mock treated or simultaneously incubated with 50 μM PD98059 or DMSO (0.1%, v/v) during the infection with CHN06 or NX0101 (105 TCID50 0.2 ml−1). At 3 d p.i., monolayers were fixed and stained for gp85 expression, which was visualized by epifluorescent microscopy. Data are representative of two independent experiments. CHN06 group, bar = 200 μm; NX0101 group, bar = 100 μm.
Figure 13
Figure 13. Immunohistochemical and western blot analysis of tumor cells from the liver of ALV-J-infected chickens.
(a) Fresh tissues were collected and fixed in 10% neutralized buffered formalin, dehydrated, embedded in paraffin wax, and then sliced into 6-μm sections. These sections were routinely stained with hematoxylin and eosin (HE), and examined microscopically. Tissues were further immunohistochemically labeled for gp85, ERK or p-ERK with JE9 monoclonal, anti-p44/42 MAPK or anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibodies, respectively. Bar = 100 μm. (b) Western blot analysis detected high expression of p-ERK proteins in myeloid leukosis (ML) samples, but not in the normal samples. Sample identification numbers are indicated above the panel as follows: T, tumor samples; C, non-tumor samples.
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