Identification And Machinism On Apoptosis Regulation By Prenylation Modification Of African Swine Fever Virus

African swine fever(ASF)is an acute and fulminating contagious disease in wild boar and domestic pigs that is caused by infection with African swine fever virus(ASFV),leading to almost 100%mortality.ASF is a category A animal infectious disease specified by the World Organization for Animal Health(OIE),and is a type of animal infectious disease that must be reported in China.ASFV was isolated and identified in China for the first time since 2018,causing a devastating blow to Chinese pig industry.Due to the limited understanding of the basic biological characteristics of ASFV and the mechanism of immune escape,there are no commercially effective vaccines and antiviral drugs.This study focused on the prenylation modification of ASFV-encoded proteins,especially the prenylation modification of pA179 L,to study the molecular mechanism of the modification of pA179 L to inhibit cell apoptosis.This study aims to explore the relationship between prenylation and apoptosis and the pathogenicity of ASFV,expanding the understanding of the basic biological characteristics of ASFV,and providing new ideas for the research of other viruses.Prenylation modification is a kind of protein post-translational modification in which a 15 C-atom farnesyl group or 20 C-atom geranylgerantl group is covalently attached to the carboxyl terminal of the protein.Hundreds of human cell proteins have been confirmed to undergo prenylation modification,and this modification abnormality is associated with a variety of human diseases.As a strict cellular parasite,viruses may also use prenylation to regulate the function of viral proteins.In order to study the relationship between prenylation modification and ASFV infection,in this study,ASFV HN/18 strain was used to infect MA104 cells,and DMSO,1?M HMG-CoA inhibitor Lovastatin,15?M farnesyltransferase were used respectively.It was found that Lovastatin,Lonafarnib and GGTI can significantly inhibit the infection and replication of ASFV,through indirect immunofluorescence and western blot analysis,indicating that the infection and replication of ASFV depend on prenylation modification.In this study,the amino acid sequences of all published ASFV-encoded proteins were downloaded from the NCBI database and the African swine fever virus database,then the candidate proteins containing prenylated modified motifs were obtained by screening their sequences.Subsequently,the Pre PS algorithm is used to predict the type and possibility of prenylation modification of these candidate proteins.The analysis found that the 4 proteins and 7 motifs encoded by ASFV is likely to be prenylated.These proteins and motifs are pA179L(CNLI),pI9R(CVIL/CVIM/CVFM),pX69R(CKCY)and pL83L(CTII/CTIL).In order to confirm that these proteins encoded by ASFV can be modified by isoprenylation,this study replaced the CASQ motif of yeast Ydj1p with CNLI,CVIL,CVIM,CVFM,CKCY,CTII,and CTIL motifs encoded by ASFV,respectively.Ydj1p plasmids expressing different prenylated motifs.Then these plasmids were transformed into Ydj1p gene-deficient yeast.Through temperature sensitivity test,it was found that CNLI,CVIL,CVIM,CTII,and CTIL motifs have extremely high levels of prenylation,and CVFM and CKCY motifs have high heterogeneity.The level of pentadienylation.Furthermore,gel mobility experiments proved that these motifs can undergo prenylation modification.Next,the Ydj1p plasmids expressing different motifs were transfected individually or together with the plasmids expressing FTase into yeast cells with double Ydj1p and FTase gene deletions,thereby identifying the prenylation modification types of the motifs encoded by different ASFVs..Subsequently,this study constructed ?-factor plasmids expressing different motifs and transformed them into MFA1/MFA2 ?-factor or MFA1/MFA2 ?-factor and Rce1 gene-deficient yeast cell lines,and passed the halo test and hybridization.Experimental and quantitative analysis methods have identified the cutting process of these motifs after prenylation modification.The results show that CVIL,CVIM,CTII and CTIL have higher cutting efficiency,CVFM motif has lower cutting efficiency,and CNLI and CKCY motifs can hardly be cleaved by Rce1.In order to further verify the prenylation modification of various protein nuclear motifs encoded by ASFV in mammalian cells,this study constructed the GFP-Kras4A-CXXX reporter system,which guided the cytoplasmic membrane of Ras protein through prenylation.Positioning to identify whether these motifs can undergo prenylation modification.Through the use of isopentenyl transferase inhibitors,combined with IFA,membrane plasmon separation and WB experiments,the types of prenylation of these motifs in mammalian cells were further distinguished.In this study,the full-length pA179L,pL83 L,pX69R and pI9R proteins were expressed.After co-immunoprecipitation,WB analysis with anti-farnesyl antibody found that these proteins encoded by ASFV can all be prenylated.Further through IFA experiments,it was found that the prenylation modification played a decisive role in the subcellular localization of these proteins.In order to explore the effect and mechanism of the prenylation modification of pA179L in inhibiting cell apoptosis,the HMG-Co A inhibitor Lovastatin,the farnesyltransferase inhibitor Lonafarnib and the geranylgeranyltransferase inhibitor GGTI-298 TFA were used,following nucleus-cytoplasm fraction,IFA and WB experiments found that the prenylated A179L-WT is mainly located in the cytoplasm,while the A179L-C176 S with prenylation blunt mutation presents a diffuse distribution in the cytoplasm and nucleus in mammalian cells.In addition,p A179 L mainly undergoes geranylgerantlation,rather than farnesylation.Next,this study analyzed the inhibitory activity of A179L-WT and A179L-C176 S on apoptosis.The results show that the A179L-WT can inhibit staurosporine-induced apoptosis,while A179L-C176 S loses the function of apoptosis supression.Furthermore,the results indicate that both A179L-WT and A179L-C176 S can interact with the swine pro-apoptotic protein BAX through the immunoprecipitation test,suggesting that the prenylation of pA179 L does not affect the protein-protein interaction.Next,through the membrane-cytoplasm fraction,it was found that the inactive mutation of isoprene significantly reduced the cell membrane localization of pA179 L.At the same time,the results of the IFA test showed that A179L-WT co-localizes with the mitochondrial marker protein COXIV,while A179L-C176 S cannot co-localize with COXIV,indicating that prenylation modification is necessary for the mitochondrial localization of pA179 L.In summary,this study found that the pA179L,pL83L,pI9R,and pX69R proteins encoded by ASFV can be prenylated,and this study confirmed that prenylation regulates its anti-apoptotic activity by directing the mitochondrial localization of pA179L protein.This study will provide new insights into the relationship between prenylation modification of ASFV-encoded protein and cell apoptosis as well as virus pathogenicity.It will also expand our understanding of the biological characteristics and protein modification of ASFV.The analysis of the ASFV proteins prenylation may shed light on elucidating the mechanisms of virus pathogenicity,and also has certain reference value on other viral researches.