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Point-Mutation Analysis of HBV Core in 5 th Helix Region which influence pgRNA Encapsidation

Point-Mutation Analysis of HBV Core in 5 th Helix Region which influence pgRNA Encapsidation. 2001. 12. 3. Jin kyung Noh Molecular Genetics Lab School of Biological Science Seoul Nat’l University. Seoul National University, Molecular Genetics Lab.

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Point-Mutation Analysis of HBV Core in 5 th Helix Region which influence pgRNA Encapsidation

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  1. Point-Mutation Analysis of HBV Core in 5th Helix Region which influence pgRNA Encapsidation 2001. 12. 3. Jin kyung Noh Molecular Genetics Lab School of Biological Science Seoul Nat’l University Seoul National University, Molecular Genetics Lab.

  2. Seoul National University, Molecular Genetics Lab. Life cycle of HBV

  3. Seoul National University, Molecular Genetics Lab. Map of the analysed mutation in the core protein. D: aspartic acid(-) A: alanine(0) K: lysine(+)

  4. Seoul National University, Molecular Genetics Lab. Core protein(monomer)

  5. Seoul National University, Molecular Genetics Lab.

  6. Seoul National University, Molecular Genetics Lab.

  7. Seoul National University, Molecular Genetics Lab.

  8. Mutant constructions of 5th helix in HBVcore ☞ Focus on charged amino acids 2. Research of virion formation Endogenous polymerase assay of cytoplasmic capsid: detection of polymerase activity within capsid ☞ ☞ Capsid formation and detection of DNA & RNA within capsid ☞ Endogenous polymerase assay of viral particles Test of trans-complementation effect with wild type core proteins Seoul National University, Molecular Genetics Lab. Experimental strategy ☞

  9. transfection immunoprecipitation cell lysis EPA reaction DNA preparation Agarose gel electrophoresis Seoul National University, Molecular Genetics Lab. Materials and Methods Endogenous polymerase assay

  10. 10ml of lysate capsid Nitrocellulose membrane Nylon membrane Western blot using HBc antibody Hybridization using HBV specific probe Seoul National University, Molecular Genetics Lab. Extraction and assay of intact viral capsids containing RNA and DNA Koschel, M., and Bruss, V. 1999. J. Virol. 73:2153-2160

  11. Seoul National University, Molecular Genetics Lab. Mutant HBV constructs used in this study

  12. C. Seoul National University, Molecular Genetics Lab. In vitro endogenous polymerase assay of point mutant core particles

  13. Seoul National University, Molecular Genetics Lab. Detection of capsids by native agarose gel electrophoresis and Western blotting

  14. D. Seoul National University, Molecular Genetics Lab. Effects of mutant core on RNA packaging, and DNA synthesis

  15. Seoul National University, Molecular Genetics Lab. Detection of total pregenomic RNA

  16. Seoul National University, Molecular Genetics Lab. In vitro endogenous DNA polymerase activity of mutant viral particles

  17. Seoul National University, Molecular Genetics Lab. In vitro endogenous DNA polymerase activity by coassembling HBV core protein.

  18. HBV core protein mutant E113D +     2070073         (87.3) Capsid formation Endogenous Polymerase Assay 95.6(±12.88) 279255.6       (93.4) viral nucleic acid in capsid Enogenous polymerase assay in virions E113A + 2724948   (72.8) intensity 82.3(±8.44) (% of WT)a   231078.5    (77.3) % of WT (standard deviation) I ntensity (% of WT) E113K + N.Db N.D N.D E117D + 2293473    (96.7) 93(±11.33) 275951.2 (92.3) E117A + 1728485      (73) 76.3(±9.77)   225062.9  (75.3) E117K + N.D N.D N.D E113A-E117A +     106003.1            (8) 14.3(±4.88) N.D R112E +    133779   (18) 20 . R127E - . . . Seoul National University, Molecular Genetics Lab. Characterization of HBV core gene mutants a WT, wild type. b N.D, not detectable.

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