The Study On The Molecular Mechanisms Of EGFR Signal Transduction By BoHV-1-infected Cells

Bovine herpesvirus 1(BoHV-1),an important pathogen causing great economic burden to the cattle industry worldwide.BoHV-1 can infect cattle but not human.However,it hasn't been reported that BoHV-1 can productively infect and kill broad-spectrum of human tumor cells,such as tumor cells from tissues of lung,breast,bone and prostate.The mechanism(s)underlying BoHV-1 replication in tumor cells is rarely reported.The epidermal growth-factor receptor(EGFR)plays an important role in the pathogenicity of various tumor cells such as lung cancer.However,the interaction between BoHV-1 infection and EGFR signaling remains unknown.Since PLC?1 and Akt,the canonical EGFR downstream signaling are all activated in BoHV-1-infected MDBK cells,here we hypothesized that BoHV-1 infection activate EGFR signaling which facilitates virus replication,and cell survival were therefore affected during the infection of lung epithelia cells A549.In this study,the effects of BoHV-1 infection had on EGFR,PLC?1,Akt and Erk1/2 signaling,were detected by Western blotting in A549 cells and MDBK cells.The results were as the following:1.A significant increase of Cleaved caspase-3 protein levels,and inhibition of cell proliferation were induced in BoHV-1-infected A549 cells.2.The levels of p-EGFR,p-PLC?1,p-Akt and p-Erk1/2 were increased.The expression levels of EGFR and Akt were significantly reduced after infection for 24,36 and 48 h in A549 cells.The protein levels of PLC?1 significantly decreased only at48 hpi,but the decreased protein levels of Erk1/2 were observed at 36 and 48 hpi.3.A significant increase of the p-EGFR appeared at 4,8,12 h and 24 hpi in BoHV-1-infected MDBK cells,the protein levels of EGFR significantly decreased only at 24 hpi.4.Relative to the DMSO control,10 ?M Gefitinib had no obvious effects on virus replication at 24 h in BoHV-1-infected A549 cells,while at 36 and 48 h the virus titers were reduced by approximately 1.4-and 1.7-logs,respectively.Time-addition assay indicated that 10 ?M Gefitinib affected the virus infection at both early and later stages of infection.10 ?M Gefitinib also significantly reduced the virus production,approximately 10-fold as detected at 24 hpi in MDBK cells.In addition,relative to the DMSO control,U73122 inhibited virus production at 36 h and 48 h in BoHV-1-infected A549 cells with titer reduced by 1-and 1.1-logs,respectively.Ly294002 and U0126 didn't show inhibitory effects in BoHV-1-infected A549 cells,but Ly294002 inhibited virus replication at 24 h in BoHV-1-infected MDBK cells.5.Relative to the DMSO control,10 ?M Gefitinib significantly inhibited the expression of p-PLC?1,but it didn't affect the levels of p-Akt at 24 h,36 h and 48 h in BoHV-1-infected A549 cells.U73122 reduced the protein levels of p-Akt but it had no effect on Erk1/2 at 36 h in BoHV-1-infected A549 cells.In summary,BoHV-1 infection led to apoptotic cell death and significant inhibition of cell proliferation,but the signaling pathways such as EGFR,Akt and Erk1/2 closely associated with cancer progression,cell proliferation and surviva,were significantly activated.The activation of EGFR contribute to virus replication in BoHV-1-infected A549 and MDBK cells.The activation of PLC?1 was partially affected by EGFR and the activation of Akt was partially regulated by PLC?1,but not EGFR.We speculated that apart from EGFR,Akt signaling was also affected by viral nucleic acids and proteins,which need further identification.The activation of Akt and Erk1/2 had no obvious effects on virus replication in A549 cells,but the activation of Akt contributed to virus replication in MDBK cells.This is the first report that Akt signaling affected BoHV-1 productive infection with cell type-specific manners.