Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

1. Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression Tsurng-Juhn Huang , Cheng-Chan Lu , Jui-Chen Tsai, Wei-Jen Yao , Xuanyong Lu , Ming-Derg Lai , Hsiao-Sheng Liu , and Ai-Li Shiau J. Biol. Chem., Vol. 280, Issue 30, 27742-27754, July 29, 2005 林建州 呂秀菱 鄧喬方 彭佳琇 陳俊良 黃思偉 郭懿瑩

2. Hepatitis B virus(HBV) • Hepatitis B is a DNA Virus of the hepadnaviridae family of viruses • C - the core protein • P - the DNA polymerase • S - the 3 polypeptides of the surface antigen • (preS1, preS2 and S - produced from alternative translation start sites). • X- X gene

3. Hepatitis B virus gene structure ~240 bp Annu. Rev. Biochem. 1987.56:651-693

4. Major S protein

6. Two Control Elements in the Hepatitis B Virus S-Promoter Are Important for Full Promoter Activity Mediated by CCAAT-Binding Factor HEPATOLOGY Vol. 29, No. 4, 1999 CLAUS-THOMAS BOCK, STEFAN KUBICKA, MICHAEL PETER MANNS, AND CHRISTIAN TRAUTWEIN

7. The biological role of M protein in the viral life cycle has been controversial ? • In vitro studies suggest that M protein is not essential for in vitro • HBV replication • Virion morphogenesis • Infectivity • IN vivo the M protein –deficient mutant can be found in patients with fulminant hepatitis

8. Effect of M protein initiation codon mutation on HBV surface gene expression Huang, T.-J. et al. J. Biol. Chem. 2005;280:27742-27754

9. To investigate the specific region within M protein involved in regulating surface gene expression. We hypothesized that the N terminus of M protein was the most likely candidate region because the main difference between M and S proteins is there N’ C’ Sint

10. pS-Luc pCMV-MHBs n + Luciferase HuH-7 cells S

11. pCMV-MHBs n HA Under natural conditions, M protein may undergo some sort of proteolytic process to generate a molecular species with electrophoretic mobility similar to that of MHBs-(1–57).

12. Quantitative analysis HA-pre-S2-(1–55) had the highest level of expression Normalized

13. To further demonstrate that the pre-S2 domain has the highest transactivation activity and that the HA tag would not affect our analysis pHAMHBs n pCMV-MHBs n HA

14. The Maximal Transactivation Region Coincides with the Pre-S2 Domain

15. ppre-S1-Luc and pS-Luc reporter gene constructs

16. M protein has no effect on pre-S1 promoter activity

17. Dose-dependent activation of the S promoter by M protein expressed by the endogenous S promoter

18. Dose-dependent activation of the S promoter by M protein expressed by the heterologous promoter

19. Summary • M protein regulates surface gene expression through the S promoter.

20. pre-S2 domain may translocate to the nucleus Energy-independent Cho EW. et al., (2001) J. Cell Sci. 114, 1115–1123. In this paper, they observations M protein may undergo proteolysis To generate a molecular species with a molecular mass close to that of MHBs-(1–57) translocates across the nuclear membrane

21. Aim:The pre-S2 domain whether fused to the N or C terminus of GFP(pEGFP-N-pre-S2 or pEGFP-C-pre-S2) and transfected into HuH-7 cells. (48 h) (Propidium iodide) 1. Regardless of whether the pre-S2 domain was fused to the N or C terminus of GFP, GFP was selectively localized within the nucleus.2. pre-S2 domain alone may be able to translocate inside the nucleus.

22. Aim:The MHBs-(1–57) domain may be released from M protein after undergoing a proteolytic process through the V8 protease cleavage site. PEST sequence:prolineglutamateserine threonine site-specific mutagenesis V8 protease cleavage site chymotrypsin cleavage site (S promoter) Schematic diagram of the M protein coding region and the putative V8 protease and chymotrypsin cleavage residues at the boundary of the pre-S2 and S domains.

23. (48 h) Mutation of the V8 protease (but not chymotrypsin) cleavage site destroys the M protein transactivation potential in HuH-7 cells and HepG2 cells.

24. HuH-7 cells(nuclear extract) (His-tagged) V8 protease site mutationabolishes the nuclear translocation ability of M protein.

25. M protein may undergo proteolysis (V8 protease cleavage site) To generate a molecular species with a molecular mass close to that of MHBs-(1–57) translocated inside the nucleaus to transactivate the S promoter

26. S promoter CAF = CCAAT adjacent factor CBF = CCAAT box-binding factor Hypothesized: transcription factors within the S promoter respond to M protein-mediated transactivation

27. The transactivation ability of M protein is not mediated through the Sp1 site the transactivating effect of M protein on the S gene promoter is not mediated through the Sp1 sites

28. The transactivation activity of the pre-S2 domain is mediated through the CCAAT box CAF may play a positive regulatory role in mediating the transactivating effect of M protein in conjunction with the CCAAT box

29. The transactivation activity of M protein is mediated through the CCAAT box

30. CAF plays a positive regulatory role in mediating the transactivating effect of M protein on the CCAAT box

31. CBF: CCAAT box binding protein M protein interacts with CBF of all the three subunits

32. 郭懿瑩 07-11-14 M protein transactivates multiple copies of the CCAAT box, CAF, or a combination of the CCAAT box and CAF in a reporter gene • The pre-S2 domain transactivated the CCAAT element. Fig.8

33. Discussion Much evidence from in vitro studies shows that M protein is not essential for HBV replication in vitro, virion morphogenesis, or infectivity. pre-S2-defective mutant can be identified (fulminant hepatitis) • Chronic HBV-infected patients indicate that expression of M protein is a marker of chronicity, implying that it is indicative of active viral replication. • Transcription transactivator (MHBst) function has been ascribed to C-terminally truncated M protein.

34. Discussion In this study : pre-S2-defective mutant that is frequently isolated from acute and chronic hepatitis patients to define a novel autoregulatory role of M protein in surface gene expression. In our study, we found that M protein can transactivate the CCAAT box of the major S promoter to regulate surface expression of L, M, and S proteins.

35. Discussion • We have found and defined a novel function of M protein. • M protein expressed within the cytoplasm undergoes a proteolytic process through which an autoregulatory domain (MHBsau) is released. • MHBsau then translocates inside the nucleus through its nuclear permeabilizing effect to interact with the CCAAT box of the S gene promoter to regulate surface gene expression.

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