Regulation Of Viral Replication By Marek's Disease Virus Through DNA Damage Response Pathway

Marek's Disease(MD),a chicken malignant T-lymphocyte proliferative neoplasm caused by Marek's Disease Virus(MDV),is one of the most serious infectious diseases of global poultry industries.Currently,evolution of virulent strains causes vaccination failures in chicken.Therefore,new vaccines and prevention strategies need to be developed.To this end,it is important to study the role of viral genes and viral pathogenesis.In addition,MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genome structures and physiological characteristics between MDV and human herpesviruses.In summary,uncovering the molecular mechanism of MDV pathogenesis and clarifying the interaction between virus and host cells is of great significance for the prevention of MD and human oncology research.The study includes the following five aspects:1.MDV infection induces DNA damageThe DNA damage response pathway(DDR)is activated in response to DNA damage caused by pathogens infection.In this study,the comet assay was used to detect virus-induced DNA damage in chicken embryo fibroblast(CEF)cells.The CEF cells infected with Md5 and CVI988 for 1,2,3 and 4 days were collected for the comet assay.It showed that MDV infection induced DNA damage.The number of cells harboring DNA damage and the extent of damage were associated with the time of infection,indicating that DNA damage may be related to replication stress caused by viral replication.Western-blot analysis showed that expression levels of downstream molecular markers of DDR pathway,p53 and p21,were increased in MDV-infected cells,indicating that virus activated the DDR pathway.Therefore,virus infection-caused DNA damage triggers activation of the DDR pathway.2.The effect of DNA damage response pathway on MDV replicationViral infection activates the DDR pathway that is one host defense mechanism against invading pathogens.In turn,viruses also either inhibit the DDR pathway to eliminate the host adverse effects on viral propagation or utilize DDR signaling factors to assist its infection.In this study,we used specific inhibitors to block ATM-,ATR-,or DNA-PK-mediated DDR pathways,respectively.VP22 was increased in CEF cells treated with ATR inhibitor VE-821,indicating that MDV replication was promoted when the ATR-Chkl pathway was blocked.In contrast,VP22 was decreased by using ATR activator hydroxyurea(HU),indicating that MDV replication was suppressed by ATR-Chk1 pathway activation.It showed that pChk1 was increased at an early stage of MDV infection.However,pChkl level in virus-infected cells was lower than control cells after infection for over 16 hours.Consistently,Etoposide(ETP)was used to activate the ATR-Chkl pathway in CEF cells and showed that ETP-mediated phosphorylation of Chkl was also attenuated by MDV infection.In addition,an increased STAT3 phosphorylation along with an decreased pChkl was observed in MDV-infected cells.These together suggest that MDV infection inhibits phosphorylation of Chkl for promoting viral replication,and the underlying mechanism of which is possibly related to the STAT3 activation.3.MDV disables ATR-Chkl pathway through activating STAT3STAT3 is an important nuclear transcription factor which plays a role in a variety of physiological processes,including embryonic development,inflammation,immunity,wound healing and tumorigenesis.CEF cells infected by Md5 or CVI988 were collected at 3,6,12,24,48 and 72 h post infection for Western-blot analysis.It was found that MDV infection elevated STAT3 phosphorylation on tyrosine 705 and serine 727 as early as 3 h post infection.As the time of infection increased,levels of pSTAT3 and STAT3 were gradually increased.Inhibition of STAT3 pathway by using the STAT3 inhibitor Stattic caused the increase of pChkl in CEF cells,and rescued Chkl phosphorylation in MDV-infected cells.By Western-blot and RT-PCR analyses,it demonstrated that viral protein VP22 and mRNA of viral genes(VP22 and pp38)were reduced in Stattic-treated cells compared to control cells.It means that MDV cannot inhibit the ATR-Chkl pathway when the STAT3 pathway is blocked,thereby viral replication is repressed.This study indicates that STAT3 is a key molecule for MDV interrupting ATR-Chk1.4.Cellular oxidative stress regulates MDV replicationReactive oxygen species(ROS)can damage biomolecules(lipids,proteins and DNA),leading to various diseases such as aging,cancer,and degenerative neuronal diseases.The oxidative stress response is related to cell transformation and oncovirus reactivation.It was found that Md5 infection enhanced ROS accumulation in CEF cells by using H2DCFDA to detect ROS.It showed that ROS was gradually increased in Md5-infected cells compared with control cells by fluorescence microscopy and flow cytometric analysis.Studies have shown that ROS as a signaling molecule activates the JAK/STAT3 pathway through upregulating NADPH oxidase activity.Therefore,the NADPH oxidase inhibitors Apocynin(APO)and Diphenyleneiodonium(DPI)were used to treat CEF cells.MDV replication was inhibited in CEF cells treated with NADPH oxidase inhibitors,which prevent the STAT3 pathway inhibiting pChk1.It confirmed that mRNA levels of viral genes VP22 and pp38 in the APO and DPI-treated cells were lower than that in control cells.It means that disruption of redox balance by inhibiting NADPH oxidase activity can repress viral replication.It speculates that MDV infection activates the STAT3 pathway through enhancement of NADPH oxidase activity by intracellular ROS accumulation,which resulting in inhibiting ATR-Chkl pathway and promoting virus proliferation.This finding provides a new insight into the role of ROS-STAT3 during MDV infection.5.Molecular detection of Marek's disease virus in red-crowned cranesMDV infects Galliformes,Strigiformes,Anseriformes and Falconiformes.The diseased red-crowned cranes at Nanjing Hongshan Zoo did necropsy,it was found that tumor nodules were detected in the whole of bodies.Large,medium and small pleomorphic lymphocytes were detected in the slices of heart and liver.This is the first time to use molecular biology diagnostic techniques to identify MDV in red-crowned cranes.By using PCR and RT-PCR,Meq and gB genes were detected in the feathers,liver,kidney,muscle and spleen from two suspected infected cranes.By homologous comparison and phylogenetic tree analysis of viral protein sequences of Meq,VP22 and L-Meq,it was confirmed that a virulent strain of MDV was found in the red-crowned crane.These findings provide evidences that MDV can infect Gruiformes and cause tumorigenesis.Since the wild cranes have not been vaccinated,it may have a potential threat on these endangered populations if infected with MDV.Meanwhile,its migratory characteristics will potentially increase risk of MDV spread.