Scerrning And Identifying Of Host Proteins Interaction With CSFV NS5B

Classical swine fever(CSF)is an important porcine infectious disease that causes significant economic losses to the swine industry.C lassical swine fever virus(CSFV)is the causative agent of CSF.In recent years,acute CSF cases have rarely been found and mild atypical CSF cases are mainly observed.However,the occurrence of atypical cases makes it more difficult on the prevention and control of CSF.Persistent infection is the main reason why it is difficult to eradicate CSF in our country.Up to now,there is no effective method for the treatment of CSF,and CSF continues to threat the swine industry.Therefore,it is very necessary to study and clarify the pathogenic mechanism of CSFV.NS5 B is an RNAdependent RNA polymerase that harbors a conserved motif GDD,which is in charge of RNA replication.The structure of pestiviral NS5 B proteins resembles a right hand with fingers,palm,and thumb domains,thus exhibiting the typical general fold of RdRp.It has been shown that NS3,NS4 A,NS4B,NS5 A,and NS5 B are sufficient for the genome replication.The interactions between NSPs(Nonstructural Proteins)and NCRs(Nonstructural Proteins)have been determined to be involved in modulation of RNA replication and translation.Whereas NS5 B protein is also likely to affect CSFV replication and transcription through interacting with related host proteins.However,the mechanism of interaction between the NS5 B protein and the host proteins involved in virus replication is not clear.In this study,the CSFV NS5 B protein was used as bait and the NS5 B gene of HCLV was cloned into the pGBKT7 vector.After testing self-activation and toxicity of the bait protein,the Y2 H Gold yeast strain transformed with p GBKT7-NS5 B was transformed into the PAM cDNA library to search for the interaction proteins of HC LV NS5 B.Finally,90 positive clones were screened and obtained by hybridization experiment using different auxotrophic media.Positive clones were screened by PC R and finally 60 positive clones were obtained and the positive clo nes were sequenced.After comparing the obtained sequence results with the database of NCBI,a total of 12 host proteins were finally confirmed and could have the interactions with CSFV NS5 B protein.Host protein GAPDH,which may be related to CSFV replication and autophagy,was selected from the previously screened host proteins as the host protein for further study.Yeast return test verification of GAPDH protein interacting with CSFV NS5 B protein Subsequently,GST-Pulldown and colocalization assay were performed to verify the interactions between GADPH protein and NS5 B protein.The results showed that host protein GAPDH is an interacting protein of HC LV NS5 B.However,the roles of GAPDHinvolvement in the mechanisms of CSFV replication and autophagy remains to be further studied.In summary,12 host proteins interacted with CSFV NS5 B protein were screened by yeast two-hybrid technique,and interaction of NS5 B protein and GAPDH protein was further identified by GST-Pulldown and colocalization assay.