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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.

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Presentation on theme: "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."— Presentation transcript:

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, , 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 Annu. Rev. Biochem : ~240 bp

4 Major S protein

5

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

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

11 HA pCMV-MHBs n 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 Normalized HA-pre-S2-(1–55) had the highest level of expression

13 pCMV-MHBs n pHAMHBs n To further demonstrate that the pre-S2 domain has the highest transactivation activity and that the HA tag would not affect our analysis 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  M protein regulates surface gene expression through the S promoter. Summary

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

21 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. 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)

22 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. Aim: The MHBs-(1–57) domain may be released from M protein after undergoing a proteolytic process through the V8 protease cleavage site. site-specific mutagenesis (S promoter) V8 protease cleavage site chymotrypsin cleavage site PEST sequence: proline glutamate serine threonine

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

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

25 M protein may undergo proteolysis 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 (V8 protease cleavage site)

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 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) 1.Chronic HBV-infected patients indicate that expression of M protein is a marker of chronicity, implying that it is indicative of active viral replication. 2.Transcription transactivator (MHBs t ) function has been ascribed to C-terminally truncated M protein.

34 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. In this study : Discussion

35 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 (MHBs au ) is released. MHBs au 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. Discussion

36 Thank you !!


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