| Summary: | 博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 89 === Abstract
The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using 32P-labeling of an introduced cAMP-dependent protein kinase (PKA) target site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22 nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunochemical study. Mutation at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. Theses mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase —surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.
During the course of 43-kDa study, two proteins with apparent molecular mass of 39-kDa and 29-kDa that contained sequence of the C-terminus of polymerase were also found in nucleocapsids using the PKA labeling method. Protease protection assay indicated that these two proteins, like the 90-kDa full-length polymerase, were encapsidated inside the nucleocapsid. However the 43-kDa protein was located outside of the nucleocapsid. These data suggest that HBV nucleocapsid contain other unreported components except polymerase, genome, and kinase.
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