Screenning And Identification Of Potential Host Interactors Of Hepatitis B Virus Polymerase

Hepatitis B Virus Polymerase (HBV Pol) is the key protein of the viral life cycle. It plays a important role in HBV genome replication and nucleocapsid assembly. Yet, few studies focus on the functions of Pol and little is known about the molecular mechainism how Pol exerts its activity. Viruses always utilize host factors in many steps of their life cycles. Hence, we sreen and identify the host interactors of Pol to investigate its function in the viral life cycle.We first extracted the HBV genome RC-DNA from the HBV-positive serum and constructed the recombinate plasmid pCDNA3.1-HBV by PCR amplification. The sequencing result indicated that this HBV belongs to genotype B, subgenotype B2 and no mutation was observed in the Pol key doamin. Then the Pol open-reading frame was amplificated from pCDNA3.1-HBV to construct the bait plasmid pGBKT7-Pol. Gal4 yeast-two hybrid system was used to screen the human liver cDNA library. Then we picked out 290 clones forβ-galactosidase assay and extracted plasmid from the positive clones. After screening out the duplicates through analysis of inserts' PCR maps and Hae III digestion maps,39 unique clones were obtained. Sequencing results showed that one of them encoded the fragment of proteasome subunit beta type 5 (PSMB5). We amplified the full length of PSMB5 CDS from HepG2 cell line cDNA and constructed the plasmids pGADT7-PSMB5 and pCDNA3.1-PSMB5-Myc. pCDN-A3.1-Pol-Flag was also constructed. In order to confirm the interaction between Pol and PSMB5 in yeast, pGBKT7-Pol was co-transformated with pGADT7-PSMB5 into yeast AH109 to reset the protein interaction. To confirm the interaction in mammalian cells, pCDNA3.1-Pol-Flag and pCDNA3.1-PSMB5-Myc were co-transfected into Hela cell line to perform co-immunoprecipitation. Both results demonstrated that Pol interact with PSMB5.In conclusion, our study first found that PSMB5 interacts with HBV Pol in yeast and confirmed this interaction by co-immunoprecipitation.This protein interaction may provide clues of the function of Pol in HBV life cycle.