| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 93 === 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.
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