Verification Of The Interaction Between HCMV Protein IE86 And Autophagy-related Protein ATG3

Background Autophagy is activated early after human cytomegalovirus(HCMV)infection but,later on,the virus blocks autophagy.HCMV interacts with cellular autophagy to regulate viral replication.However,the specific molecular mechanism by which HCMV regulates autophagy is unclear.In the previous study of our group,the interaction between MCMV M122 protein and Atg3 was detected by yeast two-hybrid technique.We hypothesized that HCMV may also regulate autophagy through the interaction of viral IE86(homologous to M122)with Atg3.Objectives To test whether HCMV protein IE86 interacts with Atg3,providing a new direction for the study of HCMV IE86 biological functions.Methods 1.Construction of the target genes cloning vectors: PCR primers were designed according to the target genes UL122 and ATG3 protein coding sequences.The corresponding target fragments were amplified by RT-PCR and recovered after electrophoresis.The target genes were inserted into p MD18 T plasmid by TA cloning technology to construct p MD18T-UL122 and p MD18T-ATG3 recombinant cloning vectors,which were verified by double enzyme digestion and sequencing identification;2.Construction of the target genes expression vectors: The target fragments were amplified by PCR and recovered after electrophoresis;the pc DNA3.1 plasmid was digested by endonucleases and recovered after electrophoresis;the target fragments were inserted into the pc DNA3.1 plasmid by In-fusion technology to construct pc DNA3.1-UL122 and pc DNA3.1-ATG3 recombinant expression vectors,which were verified by double enzyme digestion and sequencing identification;3.Expression of target proteins: The pc DNA3.1-UL122 and pc DNA3.1-ATG3 plasmids without endotoxin were extracted,and the plasmids were transfected into HEK293 T cells with lipo3000.After proteins extraction,the expression of the target proteins were detected by Western Blotting;4.Co-immunoprecipitation to verify protein-protein interaction: After the target genes were expressed in HEK293 T,the proteins were extracted and the interaction between IE86 and Atg3 was detected by co-immunoprecipitation.Results 1.The recombinant cloning vectors of p MD18T-UL122 and p MD18T-ATG3 were successfully constructed,and the enzyme digestion and sequencing were confirmed;2.The recombinant expression vectors pc DNA3.1-UL122 and pc DNA3.1-ATG3 were successfully constructed,and the enzyme digestion and sequencing were confirmed;3.The vectors pc DNA3.1-UL122 and pc DNA3.1-ATG3 can successfully express IE86 and Atg3 proteins,respectively,in HEK293 T cells;4.The Co-immunoprecipitation technique failed to confirm the interaction between IE86 and Atg3.Conclusions 1.The recombinant expression vectors pc DNA3.1-UL122 and pc DNA3.1-ATG3 for mammalian cell expression were successfully constructed,and IE86 and Atg3 proteins were successfully expressed in HEK293 T,which laid a foundation for the functional study of related proteins;2.There may be no interaction between IE86 and Atg3.HCMV may regulate autophagy through other mechanisms,which needs further exploration.