To investigate the expression of PS fusion protein and discriminate between the PS fusion RNA and protein in transfected cells, series of constructs were prepared (Fig. 3). P deficient mutant from which provide pgRNA and the other HBV proteins essential for HBV life cycle was used (Kim et al., 2004). As a first step to elucidate the significance of the PS fusion RNA for the HBV life cycle, the pHpol-ΔPS mutant was generated in which the splicing is abolished by changing the conserved nucleotide A at nt 2901 in the splice acceptor site to C (Fig. 3). This mutation induces a silent mutation on the polymerase. The PS fusion protein expressing plasmid, pPS, was constructed (Fig. 3). Within HBV genome, the region encoding the HBV surface proteins contains three in-frame starts sites that share a common termination codon.
The PS fusion protein contained majority of L protein without the N-terminal 29 amino acids of the L protein and the N-terminal 47 amino acids of the polymerase.
This PS fusion construct produces M and S protein. Therefore to exclude the possible effects by the M and S protein, the pPS-ΔMS mutant in which the AUG start codon of M and S are changed. Since it have been reported that the functional importance of the spliced L RNA in avian hepadnaviruses are similar to PS RNA, the expression of a novel DHBV protein did not appear to be connected to this. To define the PS RNA effect on HBV replication, another constructs, pPS-NP and pPS-ΔMS-NP, that could not express PS fusion protein were generated.
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Fig. 3. Schematic representation of the various mutants used in this study. HBV polymerase is provided in trans (A) and in cis (B), and the constructs to supply spliced transcripts and/or product (C) are shown. The HBV genome is shown with the relative positions of four open reading frames (not to scale). P deificient provides the core, surface and X protein, pgRNA but not polymerase. Hpol-∆PS provides
HBV DNA polymerase but did not spliced RNA; abolished sites of splicing are marked as diamond. Alternative splicing on HBV polymerase construct is indicated by dotted lines between the 5′ and 3′ splice sites. The splice junction and its deduced
amino acid are indicated at the bottom of the PS fusion construct. Asterisk represents the start codon mutation site of middle and small surface gene and/or PS fusion proteins.
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Fig. 3. Schematic representation of the various mutants used in this study.
CCC
ATG →→→→ ACGACGACG FrameshiftACG FrameshiftFrameshiftFrameshift mutationmutationmutationmutation
P deficient
ATG →→→→ ACGACGACG FrameshiftACG FrameshiftFrameshiftFrameshift mutationmutationmutationmutation
P deficient
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The RNAs isolated from HBV polymerase construct, the splicing ablated construct pHpol-ΔPS, and pPS construct transfected cells were subjected to RT-PCR. In addition to the 850-bp product from pgRNA, 450 bp DNA fragment was clearly identified from HBV polymerse-transfected cells. Both the 850 and 450 bp bands were not detected in samples from untransfected cells (Fig. 4A). Single 850 bp band was detected in pHpol-ΔPS trasnsfected cells and single 450-bp was clearly detected in pPS transfected cells (Fig. 4A). As described, immunochemical analysis of the PS fusion protein revealed that it contains both the antigenic determinants of the HBV polymerase and the surface protein (Fig. 4C). Furthermore, immunoprecipitaion with anti-TP and HBs antibodies, 43-KDa doublet protein from HBV polymerase, pPS and pPS-ΔMS transfected cells but not from pPS-NP transfected cells that PS fusion protein is not synthesized (Fig. 4B). Indirect immunofluorecence assay displayed that the expression pattern of PS and PS-ΔMS fusion protein were same the expression pattern of the protein had been shown in HBV polymerase-transfected cells and exclusively presented in the perinuclear region (Fig. 4C). Expression of the polymerase and surface protein in the splicing abrogated construct was detected as same staining pattern found other reports, which are located throughout cytoplasm.
Because PS was not translated in PS-NP transfected cells, surface antigen was stained with anti-HBs. Any proteins were not found in pPS-ΔMS-NP transfected cells. These results demonstrated that each construct was properly established to analyze the PS RNA or PS fusion protein and that the distribution of PS fusion protein was distinct from those of surface protein.
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Fig. 4. Characterizations of the surface RNA and polymerase-surface fusion protein (A) RT-PCR analysis with 29 sense primer and HBV-85 antisense primer (as presented Figure. 1.). PCR products were analyzed on agarose gels. The expected DNA fragments for the PS RNA are marked as asterisk.
HBV polymerase (lane 2), Hpol-∆PS (lane3) or PS construct (lane4) and
untransfected (lane 1) were transfected into HuH7 cells. Samples of right panel are subjected to PCR after DNase I treatment to eliminate the plasmid contaminant. (B) Immunoprecipitation of the PS protein. The cells were lysed with RIPA buffer; PS proteins were immunoprecipitated with polyclonal goat anti-HBs and then performed Western blot analysis with polyclonal rabbit anti-TP antiserum. HRP-conjugated secondary antibody and ECL was used to visualize proteins. From lane 1 to lane 4 were same as indicated (A). PS-NP (lane 5), PS-∆MS (lane 6) or PS-∆MS-NP (lane
7) were transfected to HuH7 cells. (C) Immunofluorescence assay from various constructs-transfected cells. Cells were fixed and permeabilized, and detected by confocal microscopy. Rabbit polyclonal anti-TP antibody and mouse monoclonal anti-HBs antibody was used. Anti-HBs antibody was detected with an anti-mouse fluorescein isothiocyanate-labeled antibody (green) and anti-TP was detected with an anti-rabbit rhodamine-labeled antibody (red). Transfected constructs were indicated at top of each panel.
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Fig. 4. Characterizations of the surface RNA and polymerase-surface fusion protein
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D. The PS fusion protein was partially overlapped with nuclear pore complex