Molecular Mechanism Of African Swine Fever Virus Protein A179L In Regulating Cell Apoptosis And Necroptosis

African swine fever(ASF)is an acute and highly contagious infectious disease caused by African swine fever virus(ASFV).African swine fever was first discovered in Shenyang City,Liaoning Province,China in August 2018,which had a huge impact on my country's pig industry.The virus is the only member of the African swine fever virus family African swine fever virus and the only known arbovirus DNA virus.Its genome is about 170-193 kb and contains 150-167 open reading frames(Open reading frames,ORFs),encoding 150-200 kinds of proteins,containing more than 50 kinds of structural proteins.ASFV mainly infects macrophages.The viral genome encodes a variety of proteins that can interfere with the immune response of host cells,thereby promoting virus replication and proliferation.At present,the functions of many ASFV viral proteins are not yet clear.Studying the functions of viral proteins has certain potential significance for further understanding the pathogenic mechanism of the virus and for the development of new vaccines.Necroptosis is a newly discovered method of cell death in recent years,which depends on RIPK1,RIPK3 and MLKL,while apoptosis depends on Caspase.When the death receptor is activated,RIPK1 and RIPK3 undergo phosphorylation modification and form a complex,which acts on downstream MLKL.After MLKL phosphorylation modification,oligomerization occurs and transfers from the cytoplasm to the cell membrane.MLKL oligomer has the function of ion channel.After calcium ions enter the cell,the intracellular osmotic pressure is increased,the cell membrane is damaged,the integrity is lost,and finally the cell undergoes necroptosis.The viral non-structural proteins A179L,A224L,DP71L and EP153R can inhibit cell apoptosis at the initial stage of virus infection,and provide a stable place for the virus to replicate and proliferate in cells.The viral structural protein p54 can promote cell apoptosis in the later stage of virus infection and provide conditions for the virus to spread to other healthy cells.Apoptosis is the first line of defense for host cells to restrict virus replication.When apoptosis is inhibited,necroptosis becomes the body's second line of defense against pathogen invasion.Necroptosis will cause a strong inflammatory response,which in turn activates the body's immune system to fight pathogen invasion.This thesis will explore whether there are proteins in ASFV that can regulate cell necrosis.First,we analyze the gene sequence of ASFV,design primers,and amplify the open reading frame of ASFV structural protein and non-structural protein by PCR.The amplified fragment was cloned into the eukaryotic expression vector pcDNA-flag,and the plasmid construction was identified by PCR,plasmid digestion,and gene sequencing.In the end,12 kinds of ASFV structural and non-structural proteins were successfully cloned.The constructed vector was identified by Western Blot after transfection,and it was found that the two structural proteins and three non-structural proteins of ASFV can be expressed in eukaryotic cells.To further explore the effect of ASFV-encoded protein on cell necroptosis,we transfected mouse fibroblast cell line L929 cells with the cloned plasmid,and induced necroptosis with TNF-?,Smac and ZVAD(TSZ).Western Blot results showed that the A179L gene can promote TSZ-induced phosphorylation of RIPK1,RIPK3 and MLKL,and at the same time induce the release of GAPDH into the cell culture supernatant.The results of flow cytometry stained with Sytox Green showed that A179L gene can promote TSZ-induced necroptosis.We then investigated the effect of A179L on virus-induced cell death.We first used herpes simplex virus(HSV-1)as a DNA virus to simulate the apoptosis and necroptosis induced by ASFV on the porcine intestinal epithelial cell line IPEC-DQ.Western Blot results showed that A179L can inhibit HSV-1 induced cleavage of Caspase-8 and Caspase-3,and up-regulate the phosphorylation of RIPK1 and MLKL after transfection of IPEC-DQ cells.Sytox Green staining and FITC Annexin V cell flow cytometry results show that A179L gene can inhibit herpes simplex virus-induced apoptosis and promote its induced necroptosis.We further investigated whether A179L also affects the apoptosis and necroptosis induced by Sendai virus(SeV)(RNA virus)infection.The results showed that A179L inhibited SeV-induced cleavage of Caspase-8 and Caspase-3,and up-regulated the phosphorylation of RIPK1 and MLKL.Sytox Green staining and FITC Annexin V cell flow cytometry results showed that A179L gene can inhibit Sendai virus-induced cell apoptosis and promote its induced necroptosis.We finally investigated whether A179L also affects the apoptosis and necroptosis of the human bronchial epithelial cell line NL20 induced by H1N1 influenza virus.The results showed that A179L inhibited the cleavage of Caspase-8 and Caspase-3 induced by influenza virus,and up-regulated RIPK1,RIPK3 and MLKL phosphorylation.Sytox Green staining and FITC Annexin V cell flow cytometry results showed that A179L gene can inhibit influenza virus-induced cell apoptosis and promote its induced necroptosis.These results indicate that A179L can inhibit cell apoptosis induced by DNA viruses and RNA viruses,provide a stable place for the virus to replicate and proliferate in cells,and at the same time promote TNF-? and virus-induced necroptosis,promote the body's inflammatory response,and cause Histopathological damage.