A Study On The Interaction Between Glucose-regulated Protein GRP78 And HBV PreS1

Objective: As a molecular chaperone,GRP78 has a wide range of physiological functions.It has been shown in the literature and previous work of the research group that it is involved in the regulation of one or more aspects of the life cycle of HBV virus,and initially verified that GRP78 can interact with HBV PreS1.Therefore,this article will further study the interaction between GRP78 and HBV PreS1,and preliminary study the effect of GRP78 on the replication of HBV.These studies can lay a foundation for further exploration of the role of GRP78 in the regulation of HBV virus,hoping to provide a new target for clinical antiviral therapy.Methods: ⑴Interaction between GRP78 and HBV PreS1 Peptides: The GRP78 protein was expressed in E.coli B834 and then the bacterial protein was purified by nickel ion affinity chromatography.According to the results of bioinformatics,HBV PreS1 desalting peptides(pep55,pep56,pep57,pep58,pep59,pep60)were synthesized.The interaction between GRP78 and HBV PreS1 were measured by Surface Plasmon Resonance(SPR)and Isothermal Titration Calorimetry(ITC).⑵The effect of GRP78 on HBV Virus Replication: The HepG2.2.15 and HepAD38,human hepatoma cell lines with stable expression of HBV,were infected and transfected with pAd-GRP78 and siRNA respectively.The efficiency of over-expression and silencing of GRP78 were tested by qRT-PCR and Western blot respectively.The effect of GRP78 on the level of HBV replication was detected by qRT-PCR,Southern blot and Western blot.Results: ⑴Interaction between GRP78 and HBV PreS1 Peptides: The GRP78 protein was obtained.The directly interaction between GRP78 and HBV PreS1 Peptides were detected by SPR and ITC,suggesting that pep58 and pep60 could not be fitted,the KD values of the affinity of pep55 and pep57 were around 100 nM,and the KD values of the affinity of pep56 and pep59 were less than 10 nM.⑵ The effect of GRP78 on HBV Virus Replication :The levels of mRNA and protein of GRP78 were increased in HepG2.2.15 and Hep AD38,which were infected with p Ad-GRP78.In contrast,the levels of mRNA and protein of GRP78 were decreased in HepG2.2.15 and HepAD38,which were transfected with siRNA.Over-expression of GRP78 can up-regulate the level of HBV replication in HepG2.2.15 cell line.However,the replication of HBV can be inhibited by GRP78 in HepAD38.Conclusion: These results would be helpful to further explore the molecular mechanism of the interaction between GRP78 and HBV PreS1,and lay an experimental foundation for the regulation of HBV activity by HBV PreS1 peptides inhibit the activity of GRP78.To elucidate the effect of GRP78 on HBV virus,we first selected an important part of the virus life cycle,finding that GRP78 has a certain regulatory effect on the level of HBV replication.