Interaction Between Heat Shock Protein HSP70 And Hepatitis B Virus

Objective On the basis of the interaction of HSP70 and HBV Pre S1,it is documented that HSP70 play keyroles in the process of Hepatitis B virus secretion, and it is clarified that the mechanism of the transport and secretion of HBV virus particles to the cell membrane which mediated by HSP70. Ultimately, these results will lay the foundation for theresearch and development of new antiviral drugs that aim to block the secretion of HBV.Methods1) Prokaryotic expression plasimid of p W28-HSP70 containing human HSP70 gene was constructed by PCR; The recombinant adenovirus of HSP70 was constructedand packed;The over-expression of HSP70 gene was detected in Hep G2.2.15 cells.2) Three truncated plasmids of HBV Pre S1, including of GST-Pre S1X1, GST-Pre S1X2, GST-Pre S1X3 was purified by GST affinity chromatography; Thedirectly interaction between HSP70 and GST-Pre S1X1, GST-Pre S1X2, GST-Pre S1X3 was further demonstrated by pull down test.3) The directly interactions between HSP70 and Pre S1 mutant,including GST-Pre S1Δ1-13, GST-Pre S1Δ1-44, GST-Pre S1Δ16-27,GST-Pre S1Δ21-33,GST-Pre S1Δ21-47,GST-Pre S1Δ31-47,GST-Pre S1Δ41-53, were detected by Microscale Thermophoresis(MST).4) Three mutant of HSP70, including Amino acid residues T38 A,T229A, and S300 A,were constructed by site-directed mutagenesis; ATPase activity of wild and mutant HSP70 weredetected by a colorimetric assay based on the complex formation of free phosphate and ammonium molybdate with malachite green.5) The interaction between HSP70 and HBV was dectected by q RT-PCR and western blot.6) The secretion of HBV mediated by HSP70 was analyzed by q RT-PCR, western blot and ELISA.7) Gene related to the secretion was preliminary screened by high-throughputtranscriptome sequencing.Results1) It was proved by Gene sequencing thatp W28-HSP70 and p Ad Track-TO4-HSP70 was successfully constructed;HSP70 protein was purified with about 85% purity.2) The fusion proteins including GST-Pre S1X1, GST-Pre S1X2,GST-Pre S1X3 were purified; The directly interaction between HSP70 and GST-Pre S1X1, GST-Pre S1X2, GST-Pre S1X3 was detected.3) The affinity between HSP70 and Pre S1 mutant was detected by MST, suggesting that the amino acid residues 30-45 of Pre S1 played a key role for HSP70 bingding to Pre S1.4) The HSP70 mutants were overexpressed and purified;The ATPase enzyme activity decreased sharply at p H 6.0 and 9.5 to less than 30% of the activity observed at the optimal p H;The enzyme exhibited its highest activity at 37 °C;The three mutants,T38 A, T229 A, and S300 A, exhibited much lower activity than wild type HSP70.5) HBV could increase the expression of HSP70; HSP70 could depress the expression of HBV.6) HSP70 increased the secretion of HBV.7) Some candidate factors relating to cell exocytosiswere found and waited for further exprimentsvalidation.Conclusion Amino acid residues of 30-45 of Pre S1 play a key role in the interaction HSP70 between Pre S1;The ATPase activity of HSP70 has beentested; HSP70 participates in the secretion of HBV.