Interaction Research Between YWHAB And PCV2 ORF5 Protein And Its Effect On Viral Replication

Porcine circovirus type 2(PCV2)belongs to the circovirus family of Circoviridae.It is a single-strand closed-loop closed circular DNA virus,whose genome consists of 1767-1768 nucleotides.The structure of PCV2 is icosahedral symmetry,non-encapsulated,the diameter of PCV2 is about 17 nm,PCV2 is the smallest known virus that infects mammals.PCV2 infects pigs,mainly causing Porcine circovirus disease(PCVD),causing immune damage or suppression in pigs,secondary or mixed with other pathogen infections,causing serious economic losses to the pig industry.It is currently one of the most serious pathogens in the pig industry.The elucidation of the pathogenesis of PCV2 is the fundamention for better prevention and control of the hazards in the pig industry.The study of the function of viral coding products is an important aspect to reveal the pathogenesis of PCV2.PCV2 genome contains multiple open reading frames(ORFs),and the coding products of six ORFs have been identified and functionally studied.Among them,ORF5 is a new coding product of PCV2 identified by our research team.The study found that ORF5 protein has the effects of causing host cell cycle arrest,inducing endoplasmic reticulum stress response and autophagy.The host candidate protein interacting with ORF5 was screened by yeast two-hybrid technique.14-3-3 protein beta/?(YWHAB)is one of the remarkable proteins screened by ORF5.Studies have shown that YWHAB in host cells is a multifunctional protein that interacts with hundreds of proteins in cells and plays an important role in cell cycle processes,such as cell cycle regulation,apoptosis,and cell signal transduction.So does the host cell YWHAB protein interact with the ORF5 protein? What role does it play in PCV2-infected cells? What is the impact on PCV2 replication? These are the problems that the institute is trying to solve.For this purpose,the interaction between YWHAB and PCV2 ORF5 protein and the effect of YWHAB protein on PCV2 replication were studied.The following results were obtained.(1)The host cell YWHAB protein interacts with the PCV2 ORF5 protein.Co-Immunoprecipitation(Co-IP)assay showed that YWHAB protein interacts with ORF5 protein in cells.GST pull-down assay found that YWHAB protein interacts with ORF5 protein outside the cells.After the recombinant plasmid expressing YWHAB and ORF5 proteins were transfected into the cells,the colocalization of ORF5 and YWHAB protein was detected by Confocal laser scanning microscope(CLSM).(2)The transcriptional level(mRNA)and protein level of YWHAB can be upregulated in host cells infected with PCV2 and transfected with the ORF5 expression plasmid.(3)YWHAB protein inhibits the replication of PCV2 in cells.The cell lines YWHAB protein overexpressed and interferenced were constructed,and the transcription of ORF5 and Cap gene were detected by RT-qPCR,the Cap protein level were detected by Western blot.The results showed that YWHAB inhibited the transcription of ORF5 and Cap genes,and the expression of Cap protein was also significantly reduced.The results indicate that the host cell YWHAB protein has an inhibitory effect on the replication of PCV2.(4)YWHAB protein inhibits endoplasmic reticulum stress,autophagy and apoptosis induced by PCV2.The cell lines YWHAB protein overexpressed and interferenced infect PCV2,RT-qPCR and Western blot analysis showed that YWHAB protein down-regulated the transcription and protein expression of endoplasmic reticulum stress marker proteins GRP78 and GRP94 in host cells,inhibiting endoplasmic reticulum stress response.YWHAB protein down-regulated the expression of autophagy marker proteins Beclin1 mRNA level and LC3 II protein and inhibits autophagy.Flow cytometry showed that YWHAB can reduce the ROS level of cells and decrease the cell apoptosis rate.In summary,the host cell YWHAB protein inhibits endoplasmic reticulum stress,autophagy,the ROS level of cells and apoptosis in PCV2-infected cells by interacting with PCV2 ORF5 protein,inhibiting PCV2 replication.This study clarified the role of PCV2 ORF5 protein in viral infection and provided new scientific data for the elucidation of the pathogenesis of PCV2.