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

Abstract

The hepatitis B virus surface gene consists of a single open reading frame divided into three coding regions: pre-S1, pre-S2, 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 indicated that the transactivating effect of M protein is mediated through amino acids 1-57 of M protein (the MHBs au domain), although its maximal 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 rendered M protein incapable of transactivating the S promoter but also inactivated its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrated that pre-S2 interacts with the three subunits of the CCAAT box-binding factor/nuclear factor 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 MHBs au species that will be translocated inside the nucleus, where it will interact with the CCAAT box-binding factor to regulate surface gene expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might provide a plausible explanation for hepatitis B virus-associated hepatocarcinogenesis. Hepatitis B virus (HBV)1 infection causes a wide spectrum of liver diseases, including acute hepatitis, chronic active hepatitis, cirrhosis, and hepatocellular carcinoma (1-4). HBV belongs to the hepadnavirus family, and its particle coat consists of a lipid bilayer membrane and envelope proteins. It has a partial duplex DNA genome of ϳ3.2 kb and contains four overlapping open reading frames: DNA polymerase, core protein, surface antigen, and the X gene (5). The HBV surface (envelope) gene encodes three forms of viral surface proteins (6, 7). It is divided into three parts by two internal initiation codons: the pre-S1, pre-S2, and S regions. This combination of three regions forms the large surface antigen LHBsAg or L protein. In addition, the pre-S2 and S regions form the middle surface antigen MHBsAg or M protein. The small surface antigen SHBsAg or S protein, also called major S protein, constitutes the most abundant protein of HBV envelopes. The production of these three forms of surface protein (L, M, and S) is controlled by two tandem promoters. The upstream pre-S1 promoter controls the transcription of a 2.4-kb transcript that encodes L protein only. The downstream S promoter, ϳ240 bp away, specifies transcripts with heterogeneous 5Ј termini (5), with the largest transcript e...

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Fine mapping of hepatitis B virus pre‐S deletion and its association with hepatocellular carcinoma

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“…In vitro studies suggested that M protein is not essential for viral repli- Table 4. Functional mapping of pre-S deletion mutants from chronic carriers (CC) and hepatocellular carcinoma (HCC) patients (37). It is possible that disrupting the synthesis or deleting part of the M protein would lose the enhancement and result in changing the ratio of L vs. S and accumulation of L protein in ER.…”

Hepatitis B virus pre-S/S variants in liver diseases

“…In vitro studies have suggested that M protein is not essential for viral replication, virion morphogenesis, or infectivity. Huang et al[ 40 ] defined a novel regulatory role for M protein, which may undergo a proteolytic process to generate an MHBs au (aa 1-57 of M protein) species to upregulate the transcription of S promoter. In addition, the pre-S2 region of M protein binds to pHSA (aa 3-16), but the significance of this binding is unknown[ 34 ].…”