The Molecular Mechanisms Of Cell Apoptosis Induced By Orf Virus(ORFV)119 Protein

The Orf virus(ORFV),the causative agent of contagious ecthyma(commonly known as orf),belonging to the family Poxviridae,Genus Parapoxvirus.ORFV is a duplex DNA virus,about 136.6-kbp in length,with 63.67%G+C content and 132 predicted genes.The intermediate conservative genes mainly regulate the replication,morphology and structure of the virus.But both ends is relatively not conservative genes.These genes are often associated with host selection,immune avoidance,immune regulation and viral toxicity.With the replication of virus in the target cells,the virus stimulates the host to produce a variety of cytokines and chemokines,which are involved in immune regulation,antiviral and other functions.Now,some virulence genes of ORFV have been identified,including IL-10,CBP,VEGF and interferon(IFN)resistance genes,etc.The main targets of virulence genes are cytokines and chemokines of the host,NF-?B signal pathway and apoptosis pathway,etc.The synergies of these virulence factors make ORFV have the highly immunity.ORFV 125 protein is the first apoptosis-related protein encoded by orfv in the literature.It located in the mitochondria,inhibits cell apoptosis induced by UV in vitro.It can also be combined with a series of BH3-only proteins,such as Bik,Bim,to neutralize the apoptotic activity of these proteins.The preliminary study found ORFV119 protein located in the mitochondria,can inhibit the cell proliferation and induce cell apoptosis,but the specific molecular regulation mechanism is still unclear.Therefore,this research intends to in-depth research the function of ORFV119 protein and to clarify the role of regulation the host cells apoptosis,and focuses on the specific pathways and molecular mechanisms of apoptosis This topic main source:natural science foundation of China(NO.31170147)In the following texts,we will summarize the content,results and conclusion about this research respectively.Contents:In the first chapter,CCK8 cell proliferation test,DAPI staining,TUNEL test and flow cytometry were used to detect apoptosis induced by ORFV119 protein from the different perspectives.The second chapter,Caspase activation and inhibition test,apoptosis protein chip technology,Western blot and ELISA technique were adopted to analysis the mechanism of cell apoptosis induced by ORFV119 protein.Results:Part 1:To verify ORFV119 protein induces cell apoptosis.After high expression ORFV119 protein in 293T cells,CCK8 assay was used to detect cell proliferation at different points in time.After transient transfection ORFV119 protein with two different eukaryotic expression vectors,staining with DAPI in different time,confocal laser microscope was used to observe morphological changes of nuclear.Moreover,after 24 h with high expression ORFV119 protein,dying by AnnexinV-APC and 7-AAD,flow cytometry was employed to detect cell apoptosis.Part 2:To research the mechanism of apoptosis induced by ORFV119 proteinFirstly,after 24 h and 48 h with high expression ORFV119 protein,Caspase activation kits were used to detect activation degree of the different Caspase.And the molecular mechanisms of apoptosis were analyzed.Apoptosis protein microarray was utilized to get more the change of proteins or cell factors in the process of apoptosis induced by ORFV119.After 24 h and 48 h after high expression the protein,Western blot and ELISA technique were used to detect the corresponding changes in protein level,further to clarify the molecular mechanisms of apoptosis induced by ORFV119 protein.Conclusions:This study aims to reseach the function of ORFV119 protein,its effects on cell apoptosis and related molecular mechanisms through DAPI fluorescence staining,flow cytometry,protein chip,Western blot and ELISA method.The study found that ORFV119 protein was located in the mitochondria.It can inhibit cell proliferation and induce apoptosis.Its role in inducing apoptosis are as follows.ORFV119 protein increases the expression of apoptosis proteins Bax and Bak in mitochondria.Meanwhile,mitochondrial membrane permeability is increased,which results in the release of cytochrome C and pro-apoptotic proteins Smac/Diablo.Also,ORFV119 protein degradates the anti-apoptotic proteins,Bcl-2 and cIAP-2,and activates Caspase9.After release of cytochrome C,activation of Caspase9 and Apaf-1 factor,the apoptotic body is observed,leading to the activation of downstream Caspase3 and PARP,finally apoptosis is occured.ORFV119 also directly activates Caspase8 and Caspase3,involved in extrinsic pathway that induces apoptosis.Furthermore,ORFV119 protein participates mitochondrial pathways in apoptosis through transforming BID to tBID by activated Caspase8.Our study identifies the role of ORFV119 protein in inducing apoptosis and clarifies its molecular mechanisms,laying the foundation for further exploring the funtion of unknown ORFV gene.These results help to study the mechanism of pathogeny and immunity of ORFV.And the results provide theoretical basis for prevention and control of disease that caused by ORFV.And they also provide a reference for the development of new vaccines and drugs.