臺灣博碩士論文加值系統

　　B型肝炎病毒(HBV)表面基因包含一個open reading frame分成三個區域包括preS1, preS2以及S 區域。經由從三個轉譯起始位子可分別轉譯成大型表面蛋白 ( L protein ), 中型表面蛋白 ( M protein )以及小型表面蛋白( S protein )。雖然經由研究顯示中型表面蛋白對於病毒本身的複製、型態變化及病毒的感染性並非是需要的角色。在這研究當中，我們發現在自然情形下，中型蛋白可以在轉錄層面調控表面基因的表現。中型蛋白的調控作用主要媒介S promoter上的CCAAT box，序列刪除分析指出中型蛋白對基因表現的轉化功能主要透過中型蛋白上序列第1到57個氨基酸所媒介 (MHBsau)，而且其最大的轉化活性主要位於pre-S2區域。由此結論顯示在中型蛋白pre-S2/S區域間經由V8-like蛋白酵素的切割位置經由突變不僅影響中型蛋白轉化S promoter的能力，也影響了其核轉移的能力。從免疫沈澱及免疫墨點實驗顯示pre-S2主要跟CCAAT序列上CBF/NF-Y轉錄因子的subunit互相作用，此結果定義出中型蛋白在自然情形下可經由proteolytic processs產生MHBsau蛋白片段並經由轉移到細胞核中跟CBF因子作用共同調控表面基因表現的新機制。但文獻顯示CCAAT box主要位於一些固定基因的promoter位置，這些觀察結果可能可以用來解釋HBV相關連的hepatocarcinogenesis發長成因。在另一方面，我們嘗試用過渡轉染的方式證明是否中型蛋白可能透過core跟X 基因promoter來調控其他病毒基因的表現和複製功能，從此研究中指出將中型蛋白或preS2蛋白片段連同pHBV1.2質體共同轉染到細胞中顯示表面蛋白及e蛋白的分泌成劑量增加的趨勢。以北方墨點法分析共轉染的細胞其病毒訊息核酸顯示B型肝炎病毒3.5kb pregenomic、2.4/2.1kb preS/S 訊息核酸表現也同時增加，從HepG2.2.15細胞株或以HBV2質體轉染的HuH-7細胞株也得到相似的實驗結果。進一步從luciferase報導基因活性分析當中指出中型蛋白具有可以轉化core promoter的能力。利用突變方法在core promoter顯示主要反應中型蛋白轉化的區域位於HNF4結合位置，而且此調控機制主要部分媒介於Raf-MAPK訊息傳遞路徑。為了支持這些發現，南方墨點法分析顯示增加中型蛋白表現質體的轉染會增加病毒複製中間病毒DNA產物。進一步分析中型蛋白對B型肝炎病毒的增殖作用所需的序列中，第57個氨基酸突變成其他氨基酸被用來執行轉染測試，從我們初步的結果顯示中型蛋白將第57個氨基酸從glutamate突變成除了lysin之外的其他氨基酸，清楚顯示這樣的中型突變蛋白對表面蛋白基因S promoter活性有不同程度的調控能力減弱。所以從結果顯示中型蛋白可以透過核轉移和預期promoter上轉錄因子間的相互作用增加病毒基因的表現以及複製。這些結果支持了中型蛋白在病毒基因表現及複製上具有必須的角色，並且提供一個可能的方向設計新型氨基酸序列來抑制病毒的複製，進一步應用在臨床治療上。

　Hepatitis B virus (HBV) surface gene consists of a single open reading frame divided into three coding regions: preS1, preS2, and S. By alternate translation at each of the three initiation codons, L, M, and S proteins can be synthesized. Studies have shown that M protein is not essential for viral replication, virion morphogenesis, or in vitro infectivity. In this study, we show that native M protein can regulate surface gene-expression at the transcriptional level. The regulatory effect of M protein is mediated through the CCAAT box within the S promoter. Deletion-mapping analysis indicates that the transactivating effect of M protein is mediated through amino acid 1-57 of M protein (the MHBsau domain) although its maximum transactivation activity coincides with that of the pre-S2 domain. This conclusion is supported by the fact that disruption of the putative V8 protease site at the pre-S2/S domain junction not only renders the M protein incapable of transactivating the S promoter, but also inactivates its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrate that preS2 interacts with the three subunits of CBF/NF-Y, the cognate binding protein of the CCAAT box. These results demonstrate and define a novel regulatory role of M protein, which, under natural conditions, may undergo a proteolytic process to generate an MHBsau species that will be translocated inside the nucleus, where it will interact with CBF to regulate surface gene-expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might in part provide an explanation for HBV-associated hepatocarcinogenesis. In the other part, we examine whether M protein can augment other viral gene expression and replication through C and X promoters using transient transfection method. Here we show that cotransfection of M or preS2 expression plasmid with pHBV1.2 increased the HBsAg and HBeAg secretion in a dose-dependent manner. Northern blot analysis of cotransfected cells viral mRNAs indicate that the production of HBV 3.5 kb pregenomic, 2.4/2.1 kb preS/S transcripts were also increased. Similar results were obtained when HepG2.2.15 cells or pHBV2-transfected HuH-7 cells was used. Furthermore, luciferase reporter activity analysis demonstrated the transactivating ability of M protein on core promoter. Site-specific mutagenesis of core promoter demonstrated that the site responsive to M protein’s tranactivation is localized within the HNF4 element, and this effect is partially mediated through Raf-MAPK signaling pathway. In support of these findings, Southern blot analysis of intracellular viral replicative intermediates also demonstrated that the productions of relaxed circular, double-stranded and single-stranded viral DNA were enhanced in response to the increasing amounts of transfected M protein-expression plasmid. To further characterize the sequence requirement of M protein on HBV gene activation, a series of mutants conducted from 57th amino acid of M protein were constructed and subjected to transfection assay. Our preliminary results indicated that the mutation of the 57th residue from glutamate to other residues, except lysin clearly demonstrated abolish its transactivation function on surface S promoter activity. These results indicated that M protein can augment viral gene expression and replication, through its nuclear translocation ability to interact with transcriptional factors on respective promoters. These results support the essential role of M protein in viral gene expression and replication and provide the possibility of designing novel peptide to inhibit viral replication for clinical therapeutic application.

Contents --------------- 1
誌謝-------------------- 5
Abstract (Chinese) ----- 6
Abstract (English) ----- 8
Introduction ----------- 10
Specific Aims ---------- 17
Materials and Methods--- 18
Reagents---------------- 18
Antibody List----------- 21
Methods----------------- 22
Part I
Autoregulatory Function of Hepatitis B Virus M protein on Surface Gene Expression-------------- 36
Results----------------- 37
1. M protein regulated surface-protein expression at the transcriptional level------------------------- 37
2. M protein regulated surface gene expression through the major surface promoter---------------- 37
3. Maximal transactivation region coincided with the preS2 domain-------------------------------------- 38
4. MHBsAg regulates the LHBs, MHBs and SHBs protein expression via CCAAT box
through preS2 domain---- 40
5. Nuclear translocation potential of the preS2 domain-------------------------------------------------- 41
6. Disruption of V8 protease cleavage site abolishes M protein transactivating function----------------- 42
7. M protein transactivated S promoter through the CCAAT box-------------------------------------------- 43
8. M protein regulated surface gene expression by interacting with CCAAT box-binding
proteins CBF (NF-Y)------- 45
9. Comparative analysis of preS2 and CBF factor interaction with specific CBF and adjacent CAF binding region using EMSA--------------------------------------------------------------- 47
10. M protein (preS2 domain) transactivated a multimer of the CCAAT box, CAF,
and a combination of the CCAAT and CAF------------------------------------------------------------------- 48
Discussions--------------- 49
Part II
Autoregulatory Function of Hepatitis B Virus M Protein on Viral gene expression and Replication------------------------------------------------------------------------------ 53
Results------------------ 54
1. Transfection of preS2 domain of M protein increases the surface and e protein
expression--------------- 54
2. Mutation of V8 protease cleavage site renders the M protein incapable for increasing
surface and e protein secretion------------------------------------------------------------------------- 55
3. High levels of M protein correlate with high virus gene expression----------------------------------- 55
4. M protein transactivates Core promoter, X promoter -------------------------------------------------- 56
5. M proein transactivated Core promoter through the HNF4 element--------------------------------------- 58
6. The effect of M protein on SV40, AP1, AP2, and NF-�羠 reporter genes activity----------------- 60
Discussions-------------- 62
Part III
Strategy to Design Novel Synthetic Peptide to Inhibit Viral Gene Expression----------------------------- 67
Results------------------ 68
1. Mutational analysis of M protein 57th amino acid on S promoter transactivation---------- 68
References--------------- 70
Figures------------------ 80
Fig. 1. A. Diagram of an infectious HBV virion (Dane particle)------------------------------------------ 80
B. Genomic organization of hepatitis B virus (strain adw2)------------------------------ --------------- 80
Fig. 2. A. Linear genomic structure of HBV------------------------------------------------------------- 81
B. Structural relationships among Large, Middle, and Small (Major)
surface proteins--------- 81
Fig. 3. The effect of M protein initiation-codon mutation on
HBV surface gene expression----------------------------------------------------------------------------- 82
Fig. 4. M protein transactivated S promoter------------------------------------------------------------- 84
Fig. 5. Deletion mapping of M protein reveals that the maximal
transactivating region coincided with the preS2 domain-------------------------------------------------- 86
Fig. 6. preS2 domain of M protein regulates surface gene expression------------------------------------- 88
Fig. 7. Nuclear translocation potential of preS2 domain------------------------------------------------- 90
Fig. 8. Mutation of V8 protease cleavage site abolished M protein
transactivating potential------------------------------------------------------------------------------- 91
Fig. 9. M protein transactivated the S promoter through the CCAAT box----------------------------------- 93
Fig. 10. The preS2 domain interacted with CBF complex--------------------------------------------------- 95
Fig. 11. Complex formation between preS2 domain and CBF factors---------------------------------------- 97
Fig. 12. M protein transactivated multiple copies of the CCAAT box, CAF
or a combination of the CCAAT box, and CAF in a reporter gene------------------------------------------ 99
Fig. 13. preS2 domain of M protein augments the secretion of surface
and e protein from pHBV1.2 transfected cells-------------------------------------------- --------------- 100
Fig. 14. V8 cleavage residue mutation renders the M protein incapable of activation
viral gene expression---- 101
Fig. 15. M protein or preS2 domain augments HBV gene expression and viral replication-------------- 102
Fig. 16. M protein transactivates Core and X promoter -------------------------------------------------- 104
Fig. 17. M protein transactivated the Core promoter through the HNF4 element---------------------------- 105
Fig. 18. The effect of M protein on SV-40, AP1, AP2, NF-�羠 containing promoter activity-------- 107
Fig. 19. M protein mutants on viral surface gene expression--------------------------------------------- 109
作者簡歷----------------- 110

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