Screening Of Compounds Targeting Hepatitis B Virus Core Protein Dimer Formation

Objective: The aim of this research is to develop a convenient method which faithfully indicates the formation of HBV core dimers,and establish a cell model which can be used to screencompounds targeting the core dimer formation step.Methods: Plasmids that expressing the 4 pairs of split-luciferase-HBc were constructed.The split luciferases were fused to the N terminus of HBc by long glycine-serine linkers respectively.The capability of these split-luciferase-HBcs were evaluated by co-transfection and luciferase activity assays.Functions of Rluc-HBcs were analyzed by the detection of capsid-like structure and HBV DNA replication rescue experiment.A series of Rluc-HBcs mutants were constructed to analyze the contribution of Rluc-HBcs polymers to the complemented luciferase activity.A stable cell line,named NCTP6,was established which expressed RucN-HBc and RlucC-HBc in a tetracycline regulated manner.NCTP6 was used to screen a library with 672 compounds.The antiviral activity of the candidate compounds were tested on HepAD38 and HepG2.2.15 cells.Results:1.Four pairs of split luciferase complementation systems were constructed,i.e.Firefly,Renilla,Gaussia and Nano Luc luciferase.Each luciferasewas split into two fragments at specific site and fused to the N termini of HBc by the 94G4 S linker.2.We evaluated 4 pairs of split luciferases for their capability to reflect HBc dimer formation.Split Firefly,Renilla and NanoLuc luciferase fused with HBc,when complemented,produced signals that were significantly higher than the corresponding controls by about 10 fold,20 fold and 50 fold,respectively.As for the absolute value of signal,the split-Nluc-HBcs was higher than the split-Rluc-HBcs by about 10 fold,while the split-Rluc-HBcs was higher than split-Fluc-HBcs by about 100 fold.RlucC-HBcsupported HBV DNA replication,while RlucN-HBc not in our experiments.The Rluc-HBc mutants deficient in capsid formation kept a larged part of activity of luciferase when complemented.3.Plasmid named NCTPuro was constructed,which contained a RlucN-HBc and a RlucC-HBc expressing frame driven by a TRE(Tetracycline response element)-miniCMV promoter respectively,a puromycin resistant gene,and a tTA(tetracycline-controlled transactivator)gene.Linearized NCTPuro was transfected into HEK293 cells,and 20 resistant clones were obtained by puromycin selection.The Rluc activity of clone 6 presented the best response to tetracycline among the 20 clones.4.A library containing 672 compoundswas screened using NCTP6 cells,and 42 compounds were found to inhibit the RLuc activity by over 3 fold,and 6 compounds increased the RLuc activity by over 2 foldcompared to the controls.After ruling out those with apparent cytotoxicity,39 compounds were testedin the second round screening,and 12 compounds induced a dose dependent response in Rluc activity in NCTP6 cells.In round 3 screening,4 compounds inhibited HBV DNA replication in HepAD38 cells.Further study indicated that HBV009 inhibit HBV DNA replication both in HepAD38 cells and HepG2.2.15 cell(with an IC50 5.2±0.6?M and a CC50>100?M).Conclusion:1.Anew method based on split luciferase complementation was developed by which the formation of core dimers can be detected conveniently.2.A stable cell line named NCTP6 was established which can be used to screen compounds targeting the formation of HBc dimmer.3.A library containing 672 compounds were screened by using NCTP6;HBV009 showed antiviral effect and thus deserve further study.