Determination of substituted regions between HBV and DHBV

Amino acid sequences between HBV and DHBV DNA polymerase were aligned to determine which domain would be exchanged to identify the functional domains of HBV DNA polymerase (Fig. 1). To map essential domains or motifs of HBV DNA polymerase for HBV replication, chimeric DNA polymerase of HBV and DHBV were constructed in this study (Fig. 2). DHBV DNA polymerase share 28%

identity with HBV DNA polymerase and therefore there is very little primary sequence identities between the two viral polymerases. Identical, weakly similar or non-similar sequences between two enzymes are presented as yellow, green and black, respectively (Fig. 1). The subdivided N- and C-terminus of each domain of hepadnavirus polymerase are presented as boxes in Fig. 1.

Fig. 1. Amino acid sequence alignment of HBV and DHBV DNA polymerase.

Identical, weakly similar or non-similar sequences between two enzymes are shown as yellow, green, and black, respectively. To construct chimeric DNA polymerase, each domain of hepadnavirus DNA polymerase are subdivided to N- and C-terminus and presented as various colored boxes.

1 10 20 30 40 50 61

118 130 140 150 160

(118)

176 190 200 210 220 236

(176)

235 240 250 260 270 280

(235)

293 300 310 320 330 340

(293)

351 360 370 380 390 400 411

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118 130 140 150 160

(118)

176 190 200 210 220 236

(176)

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293 300 310 320 330 340

(293)

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(351)

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(410)

118 130 140 150 160

(118)

176 190 200 210 220 236

(176)

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(235)

293 300 310 320 330 340

(293)

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(351)

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(410)

(continued)

468 480 490 500 510 528

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(585)

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(643)

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(760)

819 830 840 850 860

(819)

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(875)

468 480 490 500 510 528

(468)

527 540 550 560 570

(527)

585 590 600 610 620 630

(585)

643 650 660 670 680 690 703

(643)

702 710 720 730 740 750 762

(702)

760 770 780 790 800 810 820

(760)

819 830 840 850 860

(819)

875 880 890 900 910 920 935

(875)

468 480 490 500 510 528

(468)

527 540 550 560 570

(527)

585 590 600 610 620 630

(585)

643 650 660 670 680 690 703

(643)

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(702)

760 770 780 790 800 810 820

(760)

819 830 840 850 860

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875 880 890 900 910 920 935

(875)

B. HBV and DHBV chimeric DNA polymerases constructions

Chimeric DNA polymerases were constructed to map the essential motif of DNA polymerase that support encapsidation or replication (Fig. 2). Since HBV wt construct has longer than genome length (Kim et al., 2004), this construct could support the pgRNA and mRNA expressions and supply all proteins required for encapsidation and replication. Polymerase deficient mutant, P deficient, could express pgRNA and core, surface, and X protein except polymerase (Kim et al., 2004). This P deficient mutant construct was used to co-transfect with various DNA polymerases including chimeric DNA polymerases to map the essential motifs of DNA polymerase. For the constructions of chimeric DNA polymerases of HBV and DHBV, amino acid sequence of DNA polymerase was aligned and then four domains of hepadnavirus polymerase were subdivided into N- and C-terminus (Fig. 1).

Chimeric DNA polymerases were generated by fusion PCR techniques. Then each construct is named according the exchanged region of DHBV polymerase. Spacer region was not divided calling pDS construct. Since 3'-end sequences of HBV and DHBV may influence the polymerase expressions, DH1Cdε and DH2Cdε constructs that contain 3'-end sequences of DHBV were included.

Fig. 2. Schematic diagram of chimeric DNA polymerases construct with substituted DHBV polymerase sequence. Longer than genome length of HBV adw R9 DNA was inserted into downstream of cytomegalovirus immediate early (CMV

ATG →→→ ACG→ Frameshift mutation

P deficient

ATG →→→ ACG→ Frameshift mutation

P deficient

C. Generation of polyclonal antibody specific for each domain of HBV DNA polymerase expressed from E. coli

Although all previous attempts to generate full-length active polymerase had been failed, the expressions of separated domains were possible (Beck and Nassal, 2001). On the basis of this point, separated TP, spacer, RT and RNase H domains were expressed in MC1061 strain of E. coli. As shown in Fig. 3A, E. coli MC1061 cells were transformed with fusion constructs of each domain, induced, and produced large amounts of the TP, spacer, RT and RNase H fusion proteins with approximate molecular weight of 49, 43.5, 46, and 42 KDa, respectively (Fig. 3B). Since lager proportions of fusion proteins were present as insoluble inclusion bodies, the fusion proteins were purified from the sliced SDS-PAGE gel. Western blot analysis with specific antibodies against to GST, each fusion proteins (Fig. 3C) were expressed, as expected, but small sized protein bands were shown. Such small proteins are supposed to be degraded proteins during gel purification. Each rabbit per fusion protein was subcutaneously and intradermally immunized with gel-purified fusion proteins for 6 months with several boosts. Finally, the serum of each immunized animal exhibited a strong reactivity against fusion protein antigens at 1:32,000 dilutions. All polyclonal rabbit antiserum worked well in Western blotting, but only TP specific antisera can recognize DNA polymerase in transfected cells, not other polyclonal rabbit antiserum. This TP specific antiserum will be used in this study as shown below.

Fig. 3. The bacterial expression and the purification of GST-fused HBV polymerase domains. (A) Schematic diagram of plasmid constructs for bacterial expression. The domain of the polymerase that is expressed in E. coli is shown in relative positions on polymerase open reading frame. TP, spacer, RT and RNaseH domains are indicated. TP has His tag (hatched box). (B) The expressions of HBV DNA polymerase domains in E. coli MC1061. Purified fractions were resolved by SDS-PAGE and stained with Coomassie brilliant blue. (C) Western blot of fusion protein. The first mouse anti-GST or anti-His antibodies, secondary AP-conjugated anti-mouse antibody, and PNPP substrate were used to visualize proteins by Western blot analysis.

D. Chimeric DNA polymerases constructs cannot support the encapsidation and the replication of HBV

To identify the domains of DNA polymerase that is essential for encapsidation or replication, encapsidation assay (Fig. 4A) and endogenous polymerase assay (Fig. 4B, upper and lower panel) were performed. HuH7 cells were co-transfected with chimeric DNA polymerase constructs and P deficient mutant construct that provides pgRNA and all of HBV proteins except DNA polymerase.

HBV wt construct, pPB, which provides polymerase in cis and provides all of the requirements to support HBV replication, is used as positive control. pPB transfected cells and wt HBV polymerase and P deficient mutant co-transfected cells exhibited endogenous DNA polymerase activity and encapsidated HBV nucleic acids including pgRNA and HBV DNA. After EPA, core particles were subjected on a native agarose gel electrophoresis (Fig. 4B. upper panel) or nucleic acids in core particles were extracted and separated by 1% agarose gel electrophoresis to detect single-, double-stranded, and partially double-stranded relaxed circular forms of HBV DNA synthesis, if any (Fig. 4B. lower panel). However all chimeric DNA polymerase cannot trans-complement P deficient mutant in which pgRNA encapsidation and replication rescue of P deficient mutant were not observed.

Fig. 4. Chimeric DNA polymerase constructs cannot support HBV replications.

(A) Encapsidation assay to identify the HBV nucleic acids in situ from disrupted core particles. P deficient mutant was co-transfected with chimeric DNA polymerases construct. Fixed nucleic acids were hybridized with random-primed [³²P] labeled HBV specific probes and subjected to autoradiography. (B) Endogenous polymerase assay (EPA). Isolated core particles were supplemented with 10 µCi of α- [³²P]-dATP

(3,000 Ci/mmol) and unlabeled dNTP mix in EPA reaction buffer, electrophoresed on a 1% native agarose gel, and subjected to autoradiography (upper panel). [³² P]-labeled DNA after EPA was extracted, separated, and subjected to autoradiography (lower panel). Single-, double-stranded, and partially double-stranded relaxed circular forms of HBV DNA are marked as SS, DS, and RC, respectively. All chimeric DNA polymerases were designated according to the substituted portion of DHBV DNA polymerase. For example, when N-terminal region of terminal protein of DHBV DNA polymerase was substituted into HBV DNA polymerase, chimeric DNA polymerase was designated as DTN. Abbreviation: T, terminal protein; S, spacer; R, reverse transcriptase; H, RNaseH.

Fig. 4. Chimeric DNA polymerase constructs cannot support HBV replications.

PB PC DNA3

E. Chimeric DNA polymerase RNA expression

To investigate the reason why chimeric DNA polymerase cannot support HBV encapsidation and replication, the chimeric RNAs and chimeric polymerases expressions were analyzed. pPB, wt HBV DNA polymerase or chimeric DNA polymerase construct were transfected into the HuH7 cells respectively. Total RNA was analyzed by Northern blot analysis. Upon transfection, pgRNA was expressed by the action of the CMV promoter and subgenomic RNAs were synthesized under the control of their authentic promoters. The 3.5 Kbp pgRNA, and 2.4 and 2.1 Kbp mRNAs of surface proteins were detected from the pPB and P deficient mutant transfected cells (Fig. 5). A bit smaller RNA than pgRNA would be expected for chimeric polymerases but there were no significant size differences between pgRNA and chimeric DNA polymerase RNAs. It is possible that the size differences between pgRNA and chimeric DNA polymerase RNAs may not be as apparent in Northern blot analysis. There was another RNA band marked as asterisk between pgRNA and subgenomic RNA that may be a spliced RNA (Fig 5). In DRN construct transfected cells chimeric RNA and subgenomic RNA were very low. However, using the X gene specific probe, chimeric RNA and subgenomic RNA in DRN transfected cells were detected (data not shown) and showed similar RNA level from other constructs transfected cells indicating that this very low level might be due to the probe used to detect HBV sequences in this experiment, since the probe contain most of HBV RT sequences.

Fig. 5. HBV RNA expressions from chimeric DNA polymerase transfected HuH7 cells. Northern blot analysis was performed to detect HBV subgenomic mRNA and chimeric HBV DNA polymerase mRNA. Total RNA was extracted and separated by 1% formaldehyde gel electrophoresis, transferred to nylon membranes, hybridized with random-primed ³²P labeled HBV specific probes, and subjected to autoradiography. Chimeric HBV RNA (3.4 Kbp) and 2.1 and 2.4 Kbp mRNAs for surface protein(s) are indicated by arrows. Spliced RNAs from chimeric HBV RNA are indicated by asterisk.

HuH-7

PB Pol-def HBV pol

DTN DTC

DS DRN

DRC DHN

DH1Cde DH2Cde

3.4 Kb

2.4 Kb 2.1 Kb

28S rRNA

←

←←

F. HBV DNA polymerase expression

By immunofluorescence assay, intracellular distribution of the HBV DNA polymerase was analyzed from various polymerase constructs transfected HuH7 cells with TP-specific antibody that had been prepared in this study (Fig. 2) and endoplasmic reticulum (ER) specific antibodies, Golgi specific antibodies, or peroxisome specific antibodies. The polymerase was readily detectable in the cytoplasm of transfected HuH7cells as bright red color. The merge of the images of polymerase and ER, Golgi, or peroxisome revealed that HBV polymerase are not co-localized with these organelles and distributions of polymerase in cytoplasm differ from that of surface antigen (Fig. 6A). These results suggest that similar to DHBV, major of HBV polymerase may have another function such as pathological involvement besides the functions in virus replication and encapsidation. By immunofluorecence assay with TP specific polyclonal antiserum, HBV DNA polymerase detection from HBV wt transfected cells was possible but the expression signal was weaker than HBV polymerase only expressed cells. This result was consistent with the recent report that non-recombinant HBV polymerase from HBV replicating cells were very difficult to detect, since from which core proteins are expressed and polymerase are encapsidated in core particles (Yao et al., 2000).

During virus life cycle, viral particles containing viral proteins and nucleic acids move from the site of replication to that of virus assembly so that they have to either hijack the cytoplasmic vesicle traffic or to interact directly with the cytoskeletal

transport machinery to be transported to plasma membrane (Dohner and Sodeik, 2005). Double immuno-staining was performed to understand the relationship between HBV DNA polymerase and microtubule. HBV polymerase was not co-localized with microtubule (Fig. 5Bb). This results suggest that most of cytoplasmic HBV DNA polymerase might play another role besides the participation in encapsidation since the encapsidated core particle trafficking must utilize the host cytoskeletal system even though direct interaction have not been identified yet in HBV. By immunofluorescence assay, HBV DNA polymerase and core protein did not seem to interact directly since HBV DNA polymerase and other HBV proteins localized separately (data not shown). When DNA polymerase and surface protein were double immuno-stained with TP specific antibody and surface antigen specific antibody, respectively, these proteins seemed to be co-localized (Fig. 5Ba). This result shows that the PS (polymerase-surface) fusion protein also is expressed in HBV replicating cells. This aspect will be further explored in part II. However, chimeric DNA polymerases could not detected by immunoflorescence assay.

Fig. 6. HBV DNA polymerase expression and cellular distribution from wild type of HBV, and HBV DNA polymerase and chimeric DNA polymerase constructs transfected HuH7 cells. HuH7 cells were fixed and permeabilized, and the HBV polymerase and surface proteins were visualized by immunofluoresence microscopy. The rabbit anti-TP antibody was employed to stain DNA polymerase.

ER, Golgi and peroxisome were stained with mouse anti-calnexin, anti-58K Golgi protein and anti-catalase antibody, respectively (A). HBV wt transfected cells were immuno-stained with anti-TP and anti-HBs antibody (B, left panel). HBV DNA polymerase and microtubule were immuno-stained with anti-TP antibody and mouse α tubulin antibody (B, right panel). For microtubule and DNA polymerase,

anti-mouse fluorescein isothiocyanate antibody and anti-rabbit rhodamine-labeled secondary antibodies were used, respectively. The co-localized proteins are shown as yellow.

Fig. 6. HBV DNA polymerase expression and cellular distribution from wild type of HBV, HBV DNA polymerase, and chimeric DNA polymerase constructs transfected HuH7 cells.

HuH-7

HBVpol

ER ER ER

ER GolgiGolgiGolgiGolgi peroxisomeperoxisomeperoxisomeperoxisome

A

B

HBV wt

a b

Anti-TP Anti-HBs

Anti-TP Anti-Tubulin

HuH-7

HBVpol

ER ER ER

ER GolgiGolgiGolgiGolgi peroxisomeperoxisomeperoxisomeperoxisome

A

B

HBV wt

a b

Anti-TP Anti-HBs

Anti-TP Anti-Tubulin

G. The core particle formation in chimeric DNA polymerase constructs transfected HuH7 cells

Since chimeric DNA polymerase protein could not support HBV encapsidation and replication, core particle formation was investigated from chimeric DNA polymerase expressed cells. Cytoplasmic core particles were isolated from P deficient mutant and HBV DNA polymerase or chimeric DNA polymerase co-transfected HuH7 cells. Isolated core particles are electrophoresed on the native agarose gel and then Western blot analysis was performed using the anti-HBc antibody (Koschel et al., 2000). HBV wt, RT YMHA mutant and P deficient mutant were used as positive control and core particle were detected from these constructs transfected cells since all of them provides core proteins for core particles (Fig. 7).

Also, core particles should be detected from all of chimeric DNA polymerase expressing cells because P deficient mutant are competent for core particle formation.

But core particles were not detected from two chimeric DNA polymerase constructs, DTC and DHN, expressing cells (Fig. 7). If core particle formation is independent to the expression of DNA polymerase provided in trans, core particle formation should be same between the P deficient mutants transfected cells and the polymerase constructs and P deficient mutant co-transfected cells. However, core particle formation was considerably varied between the different chimeric DNA polymerase transfected cells. Although experimental variations may exist, the expression of DTC and DHN that have DHBV sequences at C-terminal of TP and N-terminal of RNase

H, respectively, remarkably abrogated the core particle formation (FIG. 7). In contrast, core particle formation was even enhanced by the expression of DRN that have DHBV sequence at N-terminal of RT. Since there were no significant differences in core particle formations between DH1C, DH2C, DH1Cdε, and DH2Cdε, the DHBV sequences of ε, and polyadenylation site at 3' end in DH1Cdε, and DH2Cdε did not seem to have great impact on core particle formation. From these results, it can be postulated that chimeric DNA polymerase expression may influence core particle formation through primary RNA sequences from DHBV.

Fig. 7. Core particle formation from P deficient mutant and chimeric DNA polymerase constructs co-transfected HuH7 cells. Western blot analysis to detect core particles from a native agarose gel. P deficient mutant construct that provide pgRNA and core proteins was co-transfected with chimeric HBV polymerase constructs. Isolated core particles were transferred to PVDF membranes and incubated with rabbit HBc antibody. HRP-conjugated secondary antibody and ECL were used to visualize core particles.

PB PC DNA3 HBV pol

DTN DTC

DS DRN

DRC DHN

DH1Cde DH2Cde YM

DH1C DH2C

+ + + + + + + + + + + + ^{Pol def}

PB PC DNA3 HBV pol

DTN DTC

DS DRN

DRC DHN

DH1Cde DH2Cde YM

DH1C DH2C

+ + + + + + + + + + + + ^{Pol def}

IV. DISCUSSION

In this study, polyclonal antibodies against each domains of HBV DNA polymerase were generated. Immunofluoresence assay demonstrated that TP-specific antibody is useful to detect HBV DNA polymerase expression in cells although antibodies against spacer, RT, or RNase H could not detect HBV DNA polymerase in cells. Previous reports suggested that the difficulty of generating antibody against HBV DNA polymerase may be due to the impurities of polymerase antigens during the purification procedure because, instead of the polymerase antigen, contaminants were turned out to be more immunogenic than polymerase antigen that used for immunizations (zu Putlitz et al., 1999). Also several attempts to obtain HBV DNA polymerase antibodies from animals had been unsuccessful. It might be speculated that those animals had been immunized fewer times for short periods of time. The specificity of polyclonal antiserum against TP domain may be enhanced by many boost immunization for long periods of time. The failure to generate polyclonal antibody against spacer, RT or RNase H domain might be due to a rapid degradation of polymerase fragment from its C terminus, which had been demonstrated by Rehermann et al. Also, TP specific antibody can detect polymerase-surface (PS) fusion protein, the protein product of HBV RNA splicing (demonstrated in Part II).

It had been difficult to detect HBV DNA polymerase in HBV replicating cells.

Since HBV pgRNA has unusual bicistronic structure, HBV DNA polymerase is synthesized by inefficient internal initiation of translation. Also, it had been known

that HBV DNA polymerase was degraded rapidly. Recent reports have shown that DNA polymerase of DHBV and HBV are accumulated at a detectable level in the cytoplasm (Yao et al., 2000; Cao and Tavis, 2004). Putlits et al (zu Putlitz et al., 1999) reported that HBV polymerase was predominantly localized in the cytoplasm of transfected cells regardless of the presence of other HBV proteins. Tavis groups (Cao and Tavis, 2004) demonstrated that non-encapsidated duck hepatitis B virus DNA polymerase are bound to undefined cytoplasmic structure but not to endoplasmic reticulum (Yao et al., 2000; Cao and Tavis, 2004). In this study, HBV DNA polymerase is distributed as granular patterns in the cytoplasm without evident co-localization with ER, Golgi apparatus and peroxisome (Fig. 6). These results were consistent with previously reported data which shown with DHBV and HBV. Also the detection sensitivity and the signal intensity of HBV DNA polymerase were greatly reduced in HBV replicating HuH7 cells (Fig. 6) (Cao and Tavis, 2004). These results led us to speculate that HBV DNA polymerase, possibly together with other HBV proteins, may have additional roles in pathogenesis besides for the replication of virus.

In addition to the hardship of the detection system establishment, the rapid degradation of HBV DNA polymerase makes it difficult to analyze the function or the nature of it. The known functional motif for viral replication of HBV DNA polymerase such as GLY motif for DNA priming and YMDD motif for DNA polymerase activity are also present in sequence of DHBV DNA polymerase, but previous reports demonstrated that HBV DNA polymerase cannot be replaced by

DHBV DNA polymerase structurally and enzymatically. In this study chimeric DNA polymerase has been employed to elucidate which domains of HBV DNA polymerase are important for encapsidation and replication. Loeb et al. demonstrated that chimeric avian hepadnavirus is useful to identify the requirements for avian hepadnavirus encapsidation (Ostrow and Loeb, 2002, 2004). Although HBV has simpler cis-acting requirements for encapsidation than DHBV, the domains of HBV DNA polymerase that bind with these identified cis-acting sequences remain unknown. However chimeric DNA polymerase with HBV and DHBV did not support the encapsidation and replication of HBV. According to these results, several possibilities are (1) chimeric polymerase protein cannot bind to critical cis-acting element of pgRNA for HBV encapsidation or replication, or assembled capsid particle, (2) the chimeric polymerase proteins are not expressed in co-transfected HuH7 cells, (3) the primary sequence of chimeric polymerase may influence the other HBV transcripts or HBV proteins. Therefore the expressions of chimeric DNA polymerases were tested and were not visible by immunofluorescence assay. These results suspected that the stringency of encapsidation requirement for trans-acting

문서에서 Expression and Functional Analysis of Hepatitis B Virus DNA Polymerase (페이지 39-0)