1 / 79

Novel Autoregulatory Function of HBV M Protein on Surface Gene Expression

Novel Autoregulatory Function of HBV M Protein on Surface Gene Expression. 成大醫學院 病理科 分子病理實驗室 分子診斷實驗室 呂政展. Risk Factors of Hepatocellular Carcinoma. Infection by hepatitis B and C viruses (70% caused by HBV in Taiwan ) Cirrhosis of any etiology

byrd
Download Presentation

Novel Autoregulatory Function of HBV M Protein on Surface Gene Expression

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Novel Autoregulatory Function of HBV M Protein on Surface Gene Expression 成大醫學院 病理科 分子病理實驗室 分子診斷實驗室 呂政展

  2. Risk Factors of Hepatocellular Carcinoma • Infection by hepatitis B and C viruses (70% caused by HBV in Taiwan) • Cirrhosis of any etiology • Primary hemochromatosis (excessive accumulation of iron in the liver and pancrea) • Prolonged exposure to Aflatoxin B1 (cause a specific mutation in p53)

  3. Robbin’s Pathological Basis of Disease (6th ed)

  4. Natural History of Chronic HBV Infection Science 262: 369, 1993

  5. At non-replicative stage of the natural course of HBV infection, e Ag conversion is often associated with selection of preS2-defective mutant and HBV integration • Groundglass hepatocytes, a hallmark of chronic HBV infection, within cirrhotic nodule frequently overexpress L protein. These groundglass-appearance hepatocytes harbor mutations within the preS2 regions

  6. Overexpression of LHBsAg and HCC • Expression of Hepatitis B Virus Large Envelope Polypeptide Inhibits Hepatitis B Surface Antigen Secretion in Transgenic Mice (J Virol 60: 880, 1986) • Structural and Pathological Effects of Synthesis of Hepatitis B Virus Large Envelope Polypeptide in Transgenic Mice (Proc Natl Acad Sci, USA 84: 6909, 1987) • Molecular Pathogenesis of Hepatocellular Carcinoma in Hepatitis B Virus Transgenic Mice (Cell 59: 1145, 1989)

  7. Structural and Pathological Effects of Synthesis of Hepatitis B Virus Large Envelope Polypeptide in Transgenic Mice (Proc Natl Acad Sci, USA 84: 6909, 1987) • Mouse albumin promoter-not preS1 promoter • HBsAg accumulation develop hepatocellular injury that initiates a programmed response characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy.

  8. HBx gene of hepatitis B virus induces liver cancer in transgenic mice (Nature 351: 317, 1991) • HBx gene under its own regulatory element

  9. Phylogenetic Tree of Hepadnaviruses GS: ground squirrels

  10. Different S Ag Expression Patterns during Chronic HBV Infection A.Membranous form; B, Diffuse cytoplasmic form; C. Inclusion-like form; D. Marginal form (monoclonal antibody against “a” determinant Fan et al, 2000

  11. Different S Ag Expression Patterns during Chronic HBV Infection A.Membranous form; B, Diffuse cytopplasmic form; C. Inclusion-like form; D. Marginal form (preS1 monoclonal antibody) Fan et al, 2000

  12. HBV Genomic Map @

  13. * C Terminal protein Spacer DNA polymerase RNaseH X pre-S1 -S2 S

  14. Structural Relationships Among Large, Middle, and Small (Major) Surface Proteins 100 bp pre-S1 pre-S2 S 119 55 226 S M L

  15. Differential Regulation of Synthesis of HBV Surface Proteins • One virion contains 300-400 small(S) surface protein and 40-80 middle (M) and large (L) surface proteins • S surface transcripts/Ag: L surface transcripts/Ag: = 5~10:1 • Balanced synthesis of various forms is critical for virus life cycle

  16. preS1 S promoter 3213 157 2856 833 preS1 preS2 S ATG ATG ATG Small Middle Large

  17. Puzzling Facts about preS1 and S Promoters in Reporter Gene Assay • In reporter gene assay, the isolated preS1 and S promoters have similar strength • In infected livers and and transfection experiments, the preS1 transcript is about 1/10 of S transcript

  18. Linker-Scanning of S Gene PromotersPrimer extension analysis Wt 1 2 3 4 5 6 7 8 9 10 11 12 13

  19. S promoter sequence and transcriptional factor binding sites 3050 AAGGCTCACCCCTCCACACGGCGGTATTTTGGGGTGGAGCCCTCAGGCTCAGGGCATATTG ACCACAGTGTCAACAATTCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTAC TCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAATTCCACTGCCTT CCACCAAACTCTGCAGGATCCCAGAGTCAGGGGTCTGTATCTTCCTGCTGGTGGCTCCAGTT Sp1 3111 Sp1 CBF CAF Sp1 3173 Middle S Sp1 15

  20. Linker Scanning of S promoter for Elements regulating L protein expression

  21. CCAAT-box Mutation on L protein Expression in HBV Whole and Subgenomic Fragment Circular HBV DNA

  22. Surface Gene Mutants Constructs Pre-S2 S Pre-S1 WT 2856 3213 157 1 3042 3113 * ATG ATA 2 6 59 3 3154 24 4 3194 5 * ATG AGG 5 6 2948 3090 3-1 9 63 Ls3 ACCAATCGGC GGTGACCTAA 3144 3153

  23. HBV Mutants Associated with HCC Pre-S2 S Pre-S1 WT 2856 3213 157 1 3042 3113 * ATG ATA 2(6-59)Del 2 6 59 2Int 3 3154 24 4 3194 5 * ATG AGG 5 6 2948 3090 3-1 9 63 Ls3 ACCAATCGGC GGTGACCTAA 3144 3153

  24. Western blot of HBsAg protein Pre-S2 S Pre-S1 2_6-59 2856 3213 157 * ATG ATA 2_int 2856 3213 157 2_6-59 2_int WT PreS ( 0.24X, 2.2X ) S ( 1.2X, 0.23X ) αtubulin

  25. X

  26. Clinical observations from chronically HBV-infected patients indicate that expression of M protein is a marker of chronicity, implying that expression of M protein is indicative of active viral replication (Kurai et al., 1989)

  27. Infectious hepatitis B virus variant defective in pre-S2 protein expression in a chronic carrier Virology 194:137-148, 1993

  28. From HhH-7 cells Transfected with mutated preS2 in S3 HBV virion from serum of infected patients

  29. Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis (Hepatology 26:495-499, 1997) • ﹟38-year-old surgeon was HBsAg(+) in the absence of other serological HBV markers (accidental exposure to possibly infected blood?); IgM anti-HBc, anti-HBe, ALT: 4990, 10 day later this patient dies of a hyper acute form of type B FH • About 7 weeks after the death of this patient, his mother developed a fever and asthenia. She spoke of exposure to the blood of her son after an accidental needle-stick the day before the son’s hospitalization.

  30. L(LHBs) M(MHBs) S(SHBs) S pre-S1 pre-S2 p(3A)SAg (WT) 2856 3213 157 * ATG ATA p(3A)SAg/Mint

  31. 3.0 p(3A)SAg/Mint p(3A)SAg (WT) 2.5 2.2X 2.0 Relative level L 1.5 M 1.0 S 0.5 0.23X 0 0.0 α-tubulin WT Mint WT Mint WT Mint L S M

  32. Primer extension analysis p(3A)SAg/Mint p(3A)SAg (WT) 3.0 b-gal 2.35X 2.5 2.0 LHBs Relative level 1.5 1.0 MHBs 0.5 0 0.12X 0.0 Mint WT Mint WT WT Mint SHBs L M S

  33. Sequence of the preS1 and S promoter region HNF1 Oct1 HNF3 Sp1 2721 AGGTAGTTAATCATTACTTCCAAACCAGACATTATTTACATACTCTTTGGAAGGCTGGTAT TCTATATAAGCGGGAAACCACACGTAGCGCATCATTTTGCGGGTCACCATATTCTTGGGAA CAAGAGCTACAGCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAATCTT TCTGTTCCCAATCCTCTGGGATTCTTTCCCGATCATCAGTTGGACCCTGCATTCGGAGCCA ACTCAAACAATCCAGATTGGGACTTCAACCCCGTCAAGGACGACTGGCCAGCAGCCAACCA AGTAGGAGTGGGAGCATTCGGGCCAAGGCTCACCCCTCCACACGGCGGTATTTTGGGGTGG AGCCCTCAGGCTCAGGGCATATTGACCACAGTGTCAACAATTCCTCCTCCTGCCTCCACCA ATCGGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCA GGCCATGCAGTGGAATTCCACTGCCTTCCACCAAACTCTGCAGGATCCCAGAGTCAGGGGT CTGTATCTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCT 2782 TBP 2843 Large S 2904 HNF1 2965 Sp1 3026 Sp1 NF-Y 3087 CAF Sp1 Sp1 3148 3209 Middle S 49

  34. 2438 156 HBV Luciferase reporter gene 3’ 5’ nt 2438 nt 2855 nt 3114 nt 156 ATG ATG preS2 preS1 pPreS1-Luc pS-Luc Fig 2A

  35. 1.4 1.2 1.0 Relative luciferase 0.8 activity 0.6 0.4 0.2 0.0 - - - - + ( 0.5 ) pGL3-basic ( mg ) - + + + + ( 0.5 ) pPreS1-Luc - - P(3A)S(Pst)/Sint 0.1 0.2 0.4 - 0.3 pTZ19U 0.4 0.4 0.2 Fig 2B

  36. 7 6 5 Relative luciferase activity 4 3 2 1 0 - - - - + ( 0.5 ) Vector ( mg ) - + + + + ( 0.5 ) pS-Luc - - p(3A)S(Pst)/Sint 0.1 0.2 0.4 - 0.3 pTZ19U 0.4 0.4 0.2 Fig 2C

  37. 3.0 2.5 2.0 Relative luciferase activity 1.5 1.0 0.5 0.0 - - - - + ( 0.5 ) Vector ( mg ) - + + + + ( 0.5 ) pS-Luc - - pCMV-MHBs 0.1 0.2 0.4 - pCMV-Basic 0.3 0.4 0.4 0.2 Fig 2D

  38. 7 6 5 Relative luciferase activity 4 3 2 1 0 X - - - - + pGL3-Basic ( mg ) - + + + + pS-Luc - - pCMV-preS2 0.1 0.2 0.4 - pCMV-Basic 0.3 0.4 0.4 0.2

  39. preS2 S * 55 226 ATG AGG pCMV-MHBs/Sint a.a 1- 152 pCMV-MHBs1-152 a.a 1- 76 pCMV-MHBs1-76 a.a 1- 71 pCMV-MHBs1-71 a.a 1- 57 pCMV-MHBs1-57 a.a 1- 55 pCMV-PreS21-55 pCMV-PreS21-52 a.a 1- 52 a.a 1- 48 pCMV-PreS21-48 pCMV-PreS21-47 a.a 1- 47 a.a 5 - 55 pCMV-PreS25-55 a.a 16 - 55 pCMV-PreS216-55 a.a 19 - 55 pCMV-PreS219-55 Fig. 3A

  40. 5.0 4.5 4.0 3.5 3.0 Relative luciferase activity 2.5 2.0 1.5 1.0 0.5 0.0 2 3 4 5 6 7 8 9 10 11 12 13 14 1 - - - - - - - - - - - - ( mg ) - + (0.5) pGL3-Basic - + + + + + + + + + + + + + pS-Luc (0.5) - - - - - - - pCMV-MHBs/Sint - - - - - - 0.4 - - - - - - - - - - - - - 0.4 pCMV-MHBs1-152 - - - - - - - - - - - - - 0.4 pCMV-MHBs1-76 - - - - - - - - - - - - - pCMV-MHBs1-71 0.4 - - - - - - - - - - - - - 0.4 pCMV-MHBs1-57 - - - - - - - - - - - - - 0.4 pCMV-preS21-55 - - - - - - - - - - - - - 0.4 pCMV-preS21-52 - - - - - - - - - - - - - 0.4 pCMV-preS21-48 - - - - - - - - - - - - - 0.4 pCMV-preS21-47 - - - - - - - - - - - - - 0.4 pCMV-preS25-55 - - - - - - - - - - - - - 0.4 pCMV-preS216-55 - - - - - - - - - - - - - 0.4 pCMV-preS219-55 Fig. 3B - - - - - - - - - - - - pCMV-Basic 0.4 0.4

  41. Results • Deletion mapping experiments indicate that maximal transactivating region coincides with preS2 domain

More Related