B型肝炎病毒廣泛流行於全世界，人體感染後，會導致急性肝炎、慢性肝炎、肝硬化、猛爆性肝炎以及肝癌。眾多研究已證實慢性B型肝炎是造成肝癌的原因之一。在台灣，慢性B型肝炎帶原者罹患肝癌的機率比一般人高出100倍。此外，大量的B型肝炎病毒遺傳物質及基因型C之B型肝炎病毒也都會提高肝癌的發生率。分析慢性B型肝炎病毒帶原者的血清及肝組織，發現preS1及preS2兩種突變株存在於其中。帶有preS突變株的帶原者大多年長，多半被基因型C之B型肝炎病毒感染，且發生肝硬化及肝癌的機率較高。基因轉殖小鼠的實驗已證實preS2突變株(preS2區域N端缺失以及中型表面蛋白的啟動子突變)會導致肝組織癌化。在已產生肝癌的慢性B型肝炎帶原者體內可分離出大量的preS2突變株。為了瞭解preS2區域的N端(nt.1~60)是否有關鍵性的調控因子，本篇研究中，利用特定區域突變法及兩種質體設計製作了六個突變質體(pHBV1.2/LS1~6及p(3A)SAg/LS1~6)。將突變質體利用轉移感染的技術送入HuH-7細胞株，以西方墨點法分析表面蛋白的表現，且利用引子延伸法偵測其mRNA。在pHBV1.2的系統下，多數LS突變會造成三種表面蛋白大量表現，但在p(3A)SAg的系統下只有LS2突變會使表面蛋白表現增加。綜合蛋白質及mRNA的分析，各個LS突變利用轉錄及後轉錄調控來影響表面蛋白的表現。LS2突變導致增加轉錄調控進而使得表面蛋白大量表現，顯示LS2區域也許包含一段負向調控因子。進一步以膠體電泳位移分析法證實，在LS2區域會形成HuH-7核蛋白及DNA的複合物。因此，這篇研究中，我們證實了HuH-7核蛋白可能會與LS2區域黏合，並且做為抑制子，對表面蛋白進行負轉錄調控而減少表面蛋白的表現。至於到底是哪種核蛋白參與其中，還需要更進一步地研究。

Hepatitis B Virus (HBV) infection is a well-known disease in the world which causes acute hepatitis, chronic hepatitis, cirrhosis, fulminant hepatitis, hepatocellular carcinoma (HCC), and other diseases. Numerous studies have examined the risk factors of developing hepatocellular carcinoma associated with chronic hepatitis. Studies in Taiwan have shown that HBV chronic carriers have greater than 100-fold increase of relative risk of developing HBV-associated HCC than healthy people. Furthermore, higher plasma HBV DNA levels and infection with genotype C HBV were independently (and additively) associated with increased risk of HCC among Taiwanese. The preS1 and preS2 mutants were identified in sera and liver biopsies in chronic HBV carriers. HBV carriers with preS deletion mutants were older, were more frequently infected with HBV genotype C, and had more severe liver diseases. In the transgenic mouse model, it has been shown that liver carcinogenesis can be induced by preS2 mutant with N terminal internal deletion and missense mutation in the initiation codon found in HCC patients. Furthermore, the high prevalence of preS2 mutants was isolated from chronic hepatitis B patients who had developed HCC. In this study, in order to determine if there is any critically regulatory element within N-terminal region of preS2 involved in dysregulation of surface gene expression, six linker-scanning mutants spanning nucleotide positions 1-60 were constructed by site-specific mutagenesis using pHBV1.2 and p(3A)SAg as templates. These LS mutants were transfected into HuH-7 cells, the surface protein expression were analyzed by Western blot analysis and RNA levels by primer extension. The results indicated that several LS mutants have increased expression of LHBs, MHBs and SHBs in the context of pHBV1.2 relative to wild type. However, in the context of surface gene, only one LS mutant has increased expression of surface proteins. RNA analysis indicated that increased surface protein expression does not always accompanied by concomitant elevated expression of mRNAs, suggesting that transcriptional and post-transcriptional mechanisms are involved in the regulation of surface gene expression. One of the LS mutants, i.e., LS2 mutation has resulted in the up-regulation of three forms of surface proteins and mRNAs, indicating LS2 region may harbor a negative cis-element that regulate the surface gene expression at the transcriptional level. Moreover, the electrophoretic mobility shift assay analysis demonstrated that the HuH-7 nuclear proteins form a specific protein-DNA complex composed of LS2 motif. Thus, we demonstrated that the HuH-7 nuclear proteins may bind to LS2 element to function as a repressor to negatively regulate surface gene expression at the transcriptional level. However, it remains to be determined which proteins bind to LS2 in the future.

Antonucci TK, Rutter WJ (1989): Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner. Journal of Virology 63:579-583.
Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO (2002): Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. Journal of General Virology 83:2059-2073.
Arbuthnot P, Kew M (2001): Hepatitis B virus and hepatocellular carcinoma. International Journal of Experimental Pathology 82:77-100.
Beasley RP, Hwang LY, Lin CC, Chien CS (1981): Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22707 men in Taiwan. Lancet 2:1129-1133.
Blackberg J, Kidd-Ljunggren K (2003): Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma. Journal of Medical Virology 71:18-23.
Blum HE, Liang TJ, Galun E, Wands JR (1991): Persistence of hepatitis B viral DNA after serological recovery from hepatitis B virus infection. Hepatology 14:56-62.
Blumberg BS, Alter HJ, Visnich S (1965): A "new" antigen in leukemia sera. The Journal of the American Medical Association 191:541-546.
Brechot C, Hadchouel M, Scotto J, Fonck M, Potet F, Vyas GN, Tiollais P (1981): State of Hepatitis B Virus DNA in Hepatocytes of Patients with Hepatitis B Surface Antigen-Positive and -Negative Liver Diseases. Proceedings of the National Academy of Sciences 78:3906-3910.
Bruss V, Ganem D (1991): The Role of Envelope Proteins in Hepatitis B Virus Assembly. Proceedings of the National Academy of Sciences 88:1059-1063.
Bulla GA, Siddiqui A (1989): Negative regulation of the hepatitis B virus pre-S1 promoter by internal DNA sequences. Virology 170:251-260.
Chang HK, Ting LP (1989): The surface gene promoter of the human hepatitis B virus displays a preference for differentiated hepatocytes. Virology 170:176-183.
Chen BF, Liu CJ, Jow GM, Chen PJ, Kao JH, Chen DS (2006): High prevalence and mapping of pre-S deletion in hepatitis B virus carriers with progressive liver diseases. Gastroenterology 130:1153-1168.
Chisari FV, Ferrari C (1995): Hepatitis B Virus Immunopathogenesis. Annual Review of Immunology 13:29-60.
Chisari FV, Filippi P, Buras J, McLachlan A, Popper H, Pinkert CA, Palmiter RD, Brinster RL (1987): Structural and Pathological Effects of Synthesis of Hepatitis B Virus Large Envelope Polypeptide in Transgenic Mice. Proceedings of the National Academy of Sciences 84:6909-6913.
Chisari FV, Filippi P, Mclachlan A (1986): Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. Journal of Virology 60:880-887.
Courouce-Pauty AM, PLANCON A, SOULIER PJ (1983): Distribution of HBsAg subtypes in the world. Vox Sanguinis 44:197-211.
Courtois G, Baumhueter S, Crabtree GR (1988): Purified Hepatocyte Nuclear Factor 1 Interacts with a Family of Hepatocyte-Specific Promoters. Proceedings of the National Academy of Sciences 85:7937-7941.
Dane DS, Cameron CH, Briggs M (1970): Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1:695-698.
De-Medina T, Faktor O, Shaul Y (1988): The S promoter of hepatitis B virus is regulated by positive and negative elements. Molecular and Cellular Biology 8:2449-2455.
De Groote J, Fevery J, Lepoutre L (1978): Long-term follow-up of chronic active hepatitis of moderate severity. Gut 19:510-513.
Dignani JD, Lebovitz RM, Roeder RG (1983): Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11:1475-1489.
Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, Lei HY, Su IJ (2000): Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. Journal of Gastroenterology and Hepatology 15:519-528.
Fan YF, Lu CC, Chen WC, Yao WJ, Wang HC, Chang TT, Lei HY, Shiau AL, Su IJ (2001): Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. Hepatology 33:277-286.
Fernholz D, Galle PR, Stemler M, Brunetto M, Bonino F, Will H (1993): Infectious hepatitis B virus variant defective in pre-S2 protein expression in a chronic carrier. Virology 194:137-148.
Fernholz D, Stemler M, Brunetto M, Bonino F, Will H (1991): Replicating and virion secreting hepatitis B mutant virus unable to produce preS2 protein. Journal of Hepatology 13:S102-S104.
Fridovich-Keil JL, Gudas JM, Bryan IB, Pardee AB (1991): Improved expression vectors for eukaryotic promoter/enhancer studies. Biotechniques 11:572-579.
Gabrielsen OS, Hornes E, Korsnes L, Ruet A, 0yen TB (1989): Magnetic DNA affinity purification of yeast transcription factor X-a new purification principle for the ultrarapid isolation of near homogeneous factor. Nucleic Acid Research 17:6253-6267.
Ganem D, Varmus HE (1987): The Molecular Biology of the Hepatitis B Viruses. Annual Review of Biochemistry 56:651-693.
Gunther S, Fischer L, Pult I, Sterneck M, Will H (1999): Naturally occurring variants of hepatitis B Virus. Advances in Virus Research 52:25-137.
Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich WH (1984): Large surface proteins of hepatitis B virus containing the pre-s sequence. Journal of Virology 52:396-402.
Hu KQ, Siddiqui A (1991): Regulation of the hepatitis B virus gene expression by the enhancer element I. Virology 181:721-726.
Huang TJ, Li PS (2001): Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles. Biology of Reproduction 64:163-170.
Huang TJ, Lu CC, Tsai JC, Yao WJ, Lu XY, Lai MD, Liu HS, Shiau AL (2005): Novel autoregulatory function of hepatitis B virus M protein on surface gene expression. Journal of Biological Chemistry 280:27742-27754.
Huang Z-M, Yen TSB (1993): Dysregulated surface gene expression from disrupted Hepatitis B Virus genomes. Journal of Virology 67:7032-7040.
Ian HM, Gail SH, William MW, Francoise M (1989): A DNA Cassette Containing a Trimerized SV40 Polyadenylation Signal which Efficiently Blocks Spurious Plasmid-Initiated Transcription. BioTechniques 7:276-280.
IARC (1994): IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 59. Hepatitis viruses: "IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; Hepatitis viruses." pp 54-55.
Kew MC (1981): The hepatitis-B virus and hepatocellular carcinoma. Seminars in Liver Disease 1:59-67.
Kim W, Lim SO, Kim JS, Ryu YH, Byeon JY, Kim HJ, Kim YI, Heo JS, Park YM, Jung GH (2003): Comparison of proteome between Hepatitis B Virus- and Hepatitis C Virus-associated hepatocellular carcinoma. Clinical Cancer Research 9:5493-5500.
Kuo YL, Ting LP (1997): Differential regulation of major surface promoter in hepatitis B virus. DNA and Cell Biology 16:545-557.
Lin X, Qian GS, Lu PX, Wu L, Wen YM (2001): Full length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma (vol 64, pg 299, 2001). Journal of Medical Virology 64:589-+.
Lo WY, Ting LP (1994): Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome. Journal of Virology 68:1758-1764.
Lu CC, Chen M, Ou JH, Yen TSB (1995): Key role of a CCAAT element in regulating hepatitis B virus surface protein expression. Virology 206:1155-1158.
Lu CC, Yen TSB (1996): Activation of the hepatitis B virus S promoter by transcription factor NF-Y via a CCAAT element. Virology 225:387-394.
Muravenko O, Gizatullin R, Al-Amin A, Protopopov A, Kashuba V, Zelenin A, Zabarovsky E (2000): Human ALY/BEF gene Map position 17q25.3. Chromosome Research 8:562.
Norder H, Hammas B, Lee SD, Bile K, Courouce AM, Mushahwar IK, Magnius LO (1993): Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen. Journal of General Virology 74:1341-1348.
Okamoto H, Imai M, Kametani M, al. e (1987): Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission. The Japanese Journal of Experimental Medicine 57:231-236.
Okamoto H, Tsuda F, Sakugawa H, al. e (1988): Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. Journal of General Virology 69:2575-2584.
Okuda K (1992): Hepatocellular carcinoma: recent progress. Hepatology 15:948-963.
Ou JH, Rutter WJ (1987): Regulation of secretion of the hepatitis B virus major surface antigen by the preS-1 protein. Journal of Virology 61:782-786.
Persing DH, Varmus HE, Ganem D (1986): Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide. Science 234:1388-1391.
Pollicino T, Zanetti AR, Cacciola I, Petit MA, Smedile A, Campo S, Sagliocca L, Pasquali M, Tanzi E, Longo G, Raimondo G (1997): Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis. Hepatology 26:495-499.
Radziwill G, Tucker W, Schaller H (1990): Mutational analysis of the hepatitis B virus P gene product:Domain structure and RNase H activity. Journal of Virology 64:613-620.
Raney AK, Easton AJ, McLachlan A (1994): Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter. Journal of General Virology 75:2671-2679.
Raney AK, Easton AJ, Milich DR, McLachlan A (1991a): Promoter-specific transactivation of hepatitis B virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1. Journal of Virology 65:5774-5781.
Raney AK, Le HB, McLachlan A (1992): Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. Journal of Virology 66:6912-6921.
Raney AK, McLachlan A (1995): Characterization of the hepatitis B virus large surface antigen promoter Sp1 binding site. Virology 208:399-404.
Raney AK, Milich DR, McLachlan A (1991b): Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines. Journal of Virology 65:4805-4811.
Raney AK, Zhang P, McLachlan A (1995): Regulation of transcription from the hepatitis B virus large surface antigen promoter by hepatocyte nuclear factor 3. Journal of Virology 69:3265-3272.
Redeker AG (1975): Viral hepatitis: clinical aspects. The American Journal of the Medical Sciences 270:9-16.
Ron D, Vagts AJ, Dohrman DP, Yaka R, Jiang Z, Yao L, Crabbe J, Grisel JE, Diamond I (2000): Uncoupling of beta IIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain. FASEB Journal 14:2303-2314.
Schaller H, Fischer M (1991): Transcriptional control of hepadnavirus gene expression. Current Topics in Microbiology and Immunology 168:21-39.
Shafritz DA, Kew MC (1981): Identification of integrated hepatitis B virus DNA sequences in human hepatocellular carcinomas. Hepatology 1:1-8.
Siddiqui A, Jameel S, Mapoles J (1986): Transcriptional Control Elements of Hepatitis B Surface Antigen Gene Proceedings of the National Academy of Sciences of the United States of America 83:566-570.
Stibbe W, Gerlich WH (1983): Structural relationships between minor and major proteins of hepatitis B surface antigen. Journal of Virology 46:626-629.
Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R (2000): A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. Journal of General Virology 81:67-74.
Sugauchi F, Ohno T, Orito E, Sakugawa H, Ichida T, Komatsu M, Kuramitsu T, Ueda R, Miyakawa Y, Mizokami M (2003): Influence of hepatitis B virus genotypes on the development of PreS deletions and advanced liver disease. Journal of Medical Virology 70:537-544.
Szmuness W (1978): Hepatocellular carcinoma and the hepatitis B virus. Evidence for a causal association. Progress in Medical Virology 24:40-69.
The EASL Jury (2003): EASL International Consensus Conference on Hepatitis B: 13-14 September, 2002, Geneva, Switzerland: Consensus statement (Short version). Journal of Hepatology 38:533-540.
Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H, Forster A, Rabbitts TH (1997): The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-bindinq complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO Journal 16:3145-3157.
Wang HC, Huang WY, Lai MD, Su IJ (2006): Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Science 97:683-688.
Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ (2003): Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. American Journal of Pathology 163:2441-2449.
Xu ZC, Jensen G, Yen TSB (1997): Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. Journal of Virology 71:7387-7392.
Yaka R, He DY, Phamluong K, Ron D (2003): Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1. Journal of Biological Chemistry 278:9630-9638.
Yu MW, Yeh SH, Chen PJ, Liaw YF, Lin CL, Liu CJ, Shih WL, Kao JH, Chen DS, Chen CJ (2005): Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: A prospective study in men. Journal of the National Cancer Institute 97:265-272.
Yuh CH, Ting LP (1990): The genome of hepatitis B virus contains a second enhancer: cooperation of two elements within this enhancer is required for its function. Journal of Virology 64:4281-4287.
Zaret KS, Liu JK, Dipersio CM (1990): Site-directed mutagenesis reveals a liver transcription factor essential for the albumin transcriptional enhancer. Proceedings of the National Academy of Sciences 87:5469-5473.
Zhou DX, Yen TS (1991a): The ubiquitous transcription factor Oct-1 and the liver-specific factor HNF-1 are both required to activate transcription of a hepatitis B virus promoter. Molecular and Cellular Biology 11:1353-1359.
Zhou DX, Yen TSB (1990): Differential regulation of the hepatitis B virus surface gene promoters by a second viral enhancer. Journal of Biological Chemistry 265:20731-20734.
Zhou DX, Yen TSB (1991b): The hepatitis B virus S promoter comprises A CCAAT motif and two initiation regions. Journal of Biological Chemistry 266:23416-23421.