The HBsAg was determined using ELISA kit (GENEDIA, GreenCross, Kyunggi, Korea) according to the manufacturer’s protocol.
H. Glycosylation stage analysis
The pPS-∆MS transfected HuH7 cells w harvested at 2 days post-transfection.
The PS-∆MS fusion protein was immunoprecipitated with polyclonal goat HBs
antibody. For the treatment of Endoglycosidase H (Endo H), the half of immunoprecipitated sample using anti-HBs was boiled in denaturation buffer (0.5%
SDS, 1% β-mercaptoethanol) for 10 min followed by 50 units of Endo H (New England Biolabs, Beverly, MA) treatment at 37°C for 60 min in 50mM sodium
citrate (pH 5.5) buffer containing 1% NP-40. The rest half was not treated. After boiling, reaction mixtures were subjected to SDS-PAGE and performed Western blot analysis with anti-TP antibody.
I. Northern and Southern blotting
Total RNA was extracted from transfected cells using RNAzol B (TEL-TEST INC, Friendswood, Texas). In brief, cells were lysed with 0.2 ㎖ of RNAzol B per 1×106 cells, then 20 ㎕ chloroform was added to lysate. After vigorous shaking, the cell lysate was incubated on ice for 5 min and centrifuged at 12,000 g for 15 min at
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4℃. RNA in the aqueous phase that transferred to fresh tube was precipitated by addition of equal volume of isopropanol. After centrifugation, the RNA pellet was dried and then dissolved in RNase-free distilled water. 5 µg of total RNA was denatured and electrophoresed on a 1% agarose gel containing formaldehyde and blotted onto a nylon membrane. RNA on the membranes was hybridized to a 32 P-labeled random-primed probe specific for the HBV sequence. To analyze HBV DNA synthesis by Southern blotting, core DNA was extracted, separated by agarose gel electrophoresis, and hybridized to a 32P-labelled random-priming HBV specific for the HBV sequence.
J. Endogenous polymerase assay (EPA)
Isolated core particles were incubated at 37°C for 8-12 hrs with EPA reaction buffer (50mM Tris-HCl [pH 7.5], 75mM NH4Cl, 1mM EDTA, 25mM MgCl2, 0.1%
β-meracaptoethanol, 0.5% Noniodet P-40) supplemented with 0.5mM each of dCTP,
dGTP and dTTP, and 10 µCi α-32P-dATP (3,000 Ci/mmol). 32P-labeled reaction mixtures were electrophoresed on a 1% agarose gel and subjected to autoradiography.
32P –labeled DNA was extracted following the EPA reaction and separated by 1%
agarose gel electrophoresis.
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K. RNase protection assay (RPA)
To analyze spliced RNA and mRNAs, total RNAs were isolated as described above. To prepare antisense riboprobe for RPA, part of the HBV sequence (nt 2754 to 3090) was cloned into SmaI site of pGEM3Zf(+) vector that contain approximate 60 nucleotide of vector sequences. 470 nts of radiolabeled RNA probe was synthesized from this construct using T7 RNA polymerase in vitro in the presence of [32P] UTP (specific activity, 800 Ci/mmol) and then gel purified. The RPA procedure was performed using the manufacturer′s protocol (RPA IITM, Ambion). Protected PS RNA will be 148 nts in length.
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III. RESULTS
A. Detection of the spliced RNAs and the spliced product, polymerase-surface fusion protein, from HBV polymerase expressing cells
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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B. Various spliced RNAs were detected in HBV polymerase expressing cells
Previously, it was published that defective HBV genomes were derived from heterogeneous spliced pgRNA (Gunther et al., 1997; Sommer et al., 2000). The presence of spliced RNA RNA from wt HBV polymerase transfected cells was analyzed further by RT-PCR analysis with a sense primer (HBV 23) close to the 5´
end of the polymerase gene and a set of antisense primers that covers about 90% of the surface gene. In addition to the 1.6 Kbp PCR amplified product from wt HBV polymerase RNA and product from PS fusion spliced RNA, 1.0 Kbp deleted and 1.2 Kbp deleted DNA fragments were generated from PCR with same primer set (Fig. 2).
When directly sequenced the PCR product, turned four bands were identified from 1.0 Kbp and 1.2 Kbp DNA fragments. The splice donor sites are located at nt 2447 and 2471 located close to stop codon for core protein and middle of TP domain of HBV polymerase protein, respectively (Fig. 2). And the splice acceptor sites are located at nt 2903, 282, and 489 throughout the preS1/preS/S ORF. This result demonstrated that splicing event occurs from HBV polymerase RNA and pgRNAs and HBV polymerase RNA use the same splice donor and acceptor sites with pgRNA (Fig. 2). Also splicing has occurred more frequently in HBV polymerase RNA (Fig.
1C). The following experiment will be focused on only PS fusion RNA or protein and other spliced RNA that is not translated did not analyzed further.
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Fig. 2. The detection of another spliced transcript, the determination of the splice sites and the estimation of expected protein size from the spliced RNA in HBV polymerase transfected cells. Diagram of open reading frame of HBV genome and position of PCR primers was arranged. Box denotes ORFs. RT-PCR analysis with HBV-23 sense primer and HBV-37 antisense primer (indicated above the lanes) displayed the PCR amplified fragments with the expected size of the pgRNA (1.6 Kbp) and the size of the 450 bp deleted, the 1.0 Kbp deleted, and the 1.2 Kbp deleted. Nucleotide sequence joined in the spliced RNA, splice consensus sequences and expected protein sizes derived from the spliced RNA are indicated.
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Fig. 2. The detection of another spliced transcript, the determination of the splice sites and the estimation of expected protein size from the spliced RNA in HBV polymerase transfected cells.
P frame
TPTP SpacerSpacerSpacerSpacer RTRTRTRT RNaseHRNaseHRNaseHRNaseH C Another spliced RNAAnother spliced RNA Another spliced RNA
TPTP SpacerSpacerSpacerSpacer RTRTRTRT RNaseHRNaseHRNaseHRNaseH C Another spliced RNAAnother spliced RNA Another spliced RNA
← HBV 37
→ HBV 23
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C. Distribution of the PS fusion protein was distinct from the middle and the small surface protein
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 and extensively overlapped with intermediate filament
At the onset of this study, the distribution of PS fusion protein is similar that of surface protein since it contained the majority of ORF of L protein. Also previous report presented that the retention of L protein in the endoplasmic reticulum was found to be mediated by a sequence contained within a region of 35 amino acids of the preS1 carboxy-terminal portion (Rhim and Rice, 1994). Unexpectedly, the PS protein presents in ER, ERGIC and Golgi, the well-known organelles for secretory pathway and budding site for hepadnavirus, and in peroxisome. When the PS protein was expressed, it partially co-localized with ER, Golgi and peroxisome that is reflecting the expression of M and S surface protein. These results suggested that the distribution of PS protein differ from that of M and S surface protein by abrogating translation of M and S start codon.
Current research suggested that the host cell architecture of mice was selectively altered upon infection with parvovirus minute virus (Nuesch et al., 2005).
During the viral life cycle, particles containing viral proteins and nucleic acids again move from the site of their synthesis to that of virus assembly and further to the plasma membrane so that they have to either hijack the cytoplasmic membrane traffic or they interact directly with the cytoskeletal transport machinery. In particular a variety of viruses, for example, members of Herpesviridae, Adenoviridae, Parvoviridae, Poxviridae and Baculoviridae use the microtubules and the actin
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cytoskeletions (Dohner and Sodeik, 2005). Recently current research suggested an association of Vif (human immunodeficiency virus type-1 virion infectivity factor) with the intermediate filament protein, vimentin, which are distributed on the perinuclear region around which they apparently terminate in the nuclear envelope to provide a perinuclear stable core area (Lake et al., 2003). In order to identify the exact distribution and localization of the PS protein, nuclear pore complex (NPC) and various cytoskeletons were immunostained with PS fusion protein by immunofluoresence assay. Line scanning analysis displayed that PS fusion protein is partially overlapped with NPC and extensively overlapped with vimentin.
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Fig. 5. Intracellular distribution of the PS protein. (A) Distribution of the PS fusion protein in secretory pathway related organelles. The cells were transfected with PS or PS-∆MS construct. PS construct allows the expression of M and S surface
proteins. The mouse HBs antibody was used and calnexin and
proteins. The mouse HBs antibody was used and calnexin and