To analyze the expression and function of HBV DNA polymerase, various HBV DNA polymerases were constructed. The constructs that express polymerase but did not express X protein, surface protein, or surface and X proteins were prepared to investigate the effect of other HBV proteins in the expression of HBV DNA polymerase (Fig. 1A). As referred in Part I, polyclonal antibody specific for TP domain of HBV DNA polymerase was generated. The expression of HBV polymerase in the transfected HuH7 cells was analyzed by immunocytochemistry in the presence or absence of HBV proteins. The intracellular HBV DNA polymerase was exclusively localized in the cytoplasm of transfected cells, which is consistent with the previously reported studies. Cytoplasmic localizations of HBV DNA polymerase were not affected by the expression of surface protein or X protein.
Further TP-specific polyclonal antibody used in this study also detected non-recombinant HBV polymerase in HBV wt pPB transfected cells that carry the longer than a unit length of genome (data not shown). To discriminate the expression between HBV polymerase and HBV-ΔSt that express the polymerase without the surface protein, and to identify the kinetics of both proteins, double immuno-staining performed with antiserum against TP region of HBV polymerase and α-determinant of surface protein. HBV DNA polymerase began to appear at 12 hours
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post-transfection and expression level was increased until 24 hrs post-transfection (Fig. 1B). Then, expression level was drastically decreased at 48 hrs post-transfection.
The surface proteins began to appear at 24 hours post-transfection in HBV polymerase construct-transfected cells while not in surface deficient HBV polymerase construct, HBV- ΔSt, transfected cells, which show the discrepancies in the polymerase expression patterns. HBV polymerase was expressed throughout cytoplasm (Fig. 1Ba, b). Fig. 1B-b showed typical expression pattern of the HBV polymerase and surface protein. Unexpectedly, extensively overlapped HBV DNA polymerases and surface protein in the perinuclear region (Fig. 1Bc, d) were observed in some populations of transfected cells, and became evident at 48 hrs after transfection. At this time the diversity of staining pattern might lead to be speculated that HBV polymerase and surface proteins may interact while expressed in the cells during HBV replication and, so far, there have been no evidence for an interaction between these two proteins.
To analyze further this discrepancy in the polymerase expression patterns, several attempts were applied. At first, RNA was analyzed by RT-PCR with a sense primer (HBV 29) close to the 5′ end of the pgRNA and an antisense primer (HBV
85) and the presence of spliced HBV RNA was identified. Additional PCR amplified fragment was identified that are about 450 bps shorter than fragment from PCR amplified pgRNA product. This shorter fragment represented the amplified fragment from spliced RNA (Fig. 1C). Transfected DNA was eliminated by DNaseI treatment shown in the right panel of Fig. 1A. Interestingly, HBV wt and C deficient mutant
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from which expressed pgRNAs and HBV proteins except core protein, have considerably less spliced RNA than those of various HBV polymerase constructs (Fig. 1C).
Immunoprecipitation-Western blot analysis was performed to detect the proteins in the perinuclear region which show strong co-localization signal with surface proteins from various polymerase construct transfected cells. In brief, anti-HBs antibody was used to immunoprecipitate proteins from the lysates of HBV polymerase and various HBV polymerases transfected cells, and then anti-TP antibody was used for Western blotting. Doublet proteins with approximate molecular weight of 43 KDa were detected. 43 KDa doublet bands were not observed in the surface protein deficient polymerase construct transfected cells. This result showed that the 43 KDa protein was recognized by both anti-TP and anti-HBs specific antibodies, indicating that this 43 KDa protein contained the epitopes of TP of HBV polymerase and surface protein. The natures of these proteins are explored more in detail.
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Fig. 1. Detection and characterization of a spliced transcript from various HBV polymerase gene expression constructs. (A) Schematic diagram of the HBV polymerase gene expression constructs. Four ORFs of HBV are shown at the top.
Plasmid HBV-∆St, HBV-∆X and HBV-∆StX mutants were constructed from pHBV
pol by introducing stop codon at 15amino acid of S surface gene, ACG at AUG start codon of X gene, and both, respectively. (B) Different expression pattern (as a, b, c or d indicated above picture) of various proteins in HBV polymerase transfected cells.
The HBV pol, HBV polymerase transfected HuH7 cells; and the HBV-ΔSt, surface deficient mutant-transfected HuH7 cells were detected by immunofluoresence microscopy. Anti-TP antibody was a rabbit polyclonal antibody and anti-HBs antibody was a mouse monoclonal antibody. Time points were indicated at top panel.
(C) Primer position for PCR on the pgRNA and RT-PCR analysis from RNA of the transfected cell with HBV-29 sense primer and HBV-85 antisense primer (as arrows indicated above the lanes) are shown. The signals with the expected size for pgRNA are marked as diamonds, 450 bp shorter DNA bands that are from spliced RNAs are marked as asterisk. RT-PCR fragment of the RNA from HBV wt (lane 2), C deficient mutant, (lane 3), HBV polymerase (lane 4), the S deficient mutant, HBV-∆St (lane 5), the X deficient mutant, HBV-∆X (lane 6), the S and X deficient mutant, HBV-∆StX
(lane 6), or mock (lane 7)- transfected cells. (D) Immunoprecipitation of the spliced product, the PS fusion protein from various polymerase transfected cells. The lysates were immunoprecipitated with anti-HBs or anti-TP (top and bottom of C panel) antibodies and detected by Western analysis with anti-TP.
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Fig. 1. Detection and characterization of a spliced HBV transcript and spliced protein from various HBV polymerases construct transfected cells.
1
TP SpacerSpacerSpacerSpacer RTRTRTRT RNaseHRNaseHRNaseHRNaseH C
TP SpacerSpacerSpacerSpacer RTRTRTRT RNaseHRNaseHRNaseHRNaseH C
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(continued)
4343 4343kDakDakDakDa HuH
******* Heavy chainHeavy chainHeavy chainHeavy chain
C
******* Heavy chainHeavy chainHeavy chainHeavy chain 4343
4343kDakDakDakDa HuH
******* Heavy chainHeavy chainHeavy chainHeavy chain
C
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