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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 107 to 1011 virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA–transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. Conclusion: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV–host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs. (HEPATOLOGY 2010)

Hepatitis B virus (HBV) is a widespread pathogen responsible for acute and chronic hepatitis and is a causative factor of hepatocellular carcinoma (HCC).1 HBV is a small-enveloped noncytopathic virus containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells.2 During replication, the viral polymerase reverse-transcribes the pregenomic RNA to DNA using deoxyribonucleotide triphosphates (dNTPs). HBV preferentially replicates in nondividing cells,3 in which the concentration of dNTP is low, which raised the question whether dNTP concentration is adequate to support viral yield. The level of dNTPs in a nondividing adult liver cell is <0.4 μM.4 The Michaelis constant (Km) of the viral polymerase at a dNTP concentration of 0.4 μM is only 62%, whereas a 4 μM concentration gives 93.3% activity.5 Furthermore, one hepatocyte produces 50-300 hepatitis B virions per day,6 and because the HBV genome is approximately 3.2 kilobases, between 3 × 105 to 2 × 106 dNTPs are consumed per day in this process. Considering cell volume as 500 fL,7 a resting cell contains approximately 1.2 × 105 dNTP molecules. Thus, the total amount of dNTPs used for HBV production per day exceeds the amount found in a nondividing hepatocyte. Because HBV does not activate the cell cycle upon infection,8 an alternate mechanism must be used by the virus to activate dNTP production in the nondividing cells. The viral need for dNTPs led us to investigate the regulation of dNTP synthesis in HBV-infected cells.

The key enzyme responsible for de novo dNTP synthesis is ribonucleotide reductase (RNR), which is composed of R1 and R2 subunits.9 While the R1 subunit is expressed in quiescent cells, although at a low level, the R2 subunit expression is silenced.10 Here, we report that HBV increases the dNTP pool for effective viral production in quiescent cells by directly targeting the R2 gene to induce unscheduled R2 expression without affecting cell cycle progression. We further show that hepatitis B x protein (HBx), a regulatory protein of HBV, is sufficient for R2 induction by blocking the access of regulatory factor x1 (Rfx1), a repressor of the R2 gene.11

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Tissue Culture, Treatments, and Reagents.

HepG2, HepG2.2.15, HEK293T, and NIH-3T3 cells were grown as described.12 For RNR inhibition, cells were treated with 1.5 mM hydroxyurea (HU; Sigma). [Methyl-3H]thymidine was from Amersham Bioscience (TRK686, 80 Ci/mmol, 1 mCi/mL). For lentivector infections, HepG2 cells were seeded and treated with dimethyl sulfoxide (DMSO) 1 week prior to infection. Lentivirions were prepared fresh as described below, and virion-containing medium was used to transduce the HepG2 cells. The cells were washed six times in phosphate-buffered saline (PBS) 12-24 hours after infection, and 2% DMSO-containing medium was added to the cells.

[3H]Thymidine Incorporation Assay.

Cells were incubated in fresh medium containing [3H]thymidine, 7.5 μCi/well in a 24-well plate, for 4 hours. Cells were washed and stored at −80°C for at least 1 hour. Cells were then resuspended in 150 μL PBS and transferred to a 96-well plate. Using a matrix automatic reader (Micromate 196 Harvester, Packard) and a Matrix 96 beta counter (Packard) for 96-well plates, [3H]thymidine incorporation values were obtained.

[3H]Thymidine Uptake Assay.

Cells were labeled as above but only for 25 minutes. Cells were washed four times in PBS, collected in 100 μL PBS, added directly to scintillation liquid (Ultima Gold, PerkinElmer), and counted in a TRI CARB 1500 (Packard) scintillation counter.

RNA Extraction and Analysis.

RNA extraction was performed using TRI-reagent (Molecular Research Center Inc.) using standard protocols. For first-strand synthesis, 0.5 μg of RNA was taken for each sample using iScript kit (Roche). For polymerase chain reaction (PCR), 2 μL of the reverse-transcription product was taken and together with specific primers was amplified in PCR. See Supporting Information for list of primer sequences. Real-time PCR was performed by using the LightCycler 480 (Roche, Gipf-Oberfrick, Switzerland). Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA levels.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) was performed as described.11

Western Blotting.

Western blotting (protein immunoblotting) was performed as previously described.11, 13 Proteins were detected with the following antibodies: goat anti-R2 (N-18: SC-10844; Santa Cruz Biotechnology, Santa Cruz, CA), mouse antibody to hepatitis B core antigen (anti-HBcAg),13 and rabbit anti-Rfx1 polyclonal antibodies.11 The blots were then reacted with horseradish peroxidase–conjugated secondary antibody (Jackson), and enhanced chemiluminescence (ECL) detection was performed using EZ-ECL (Biological Industries).

Primary Mouse Hepatocytes.

Isolation of primary mouse hepatocytes was done according to the method of Moldeus et al.14 The method is based on collagenase digestion and separation of liver parenchymal cells.

Mice—Hydrodynamic Injection.

Hydrodynamic injection was performed as described.15 In brief, 7-week-old female BALB/C mice were injected with 1.5 mL of normal saline (0.9% NaCl) by using the high-pressure hydrodynamic method containing either a 1.3 HBV wild-type plasmid or a control plasmid. Mice were sacrificed, and livers were harvested after 2 days.

Flow Cytometry (Fluorescence-Activated Cell Sorting) Analysis.

As described,16 cells were collected and analyzed by FACSort (Becton Dickinson) fluorescence-activated cell sorting (FACS).

Bromodeoxyuridine.

Bromodeoxyuridine (BrdU) incorporation was performed as described in Beisker et al.17

Preparation of Lentiviral Transducing Particles.

HEK293T cells were seeded in 9-cm dishes and transfected with the three constructs of the lenti-system: 10 μg lenti expression vector, 7.5 μg packaging vector (cytomegalovirus delta-R8.9), and 2.5 μg envelope vector (VSV-G [vesicular stomatitis virus glycoprotein]).18 Medium was replaced 7-8 hours after transfection, using 4.5 mL to get a high viral concentration.

HBV Virions Production and Analysis.

For viral production HepG2 2.2.15 cells were grown with 2.5% DMSO for 1 week, the medium then was changed to medium containing 2.5% DMSO and 1% serum, with or without 1 mM HU. After a week the medium was collected, and centrifuged in a Sorvall SS34 rotor, at 34,633g RPM, for 30 minutes at 4°C. The cleared supernatant was ultracentrifuged at 140,000g, 16 hours at 4°C to concentrate the virions. The pellet containing virions was resuspended in 100 μL PBS (×300 concentration-fold). For protein analysis, 10 and 20 μL of the sample was resuspended with 1 mM dithiothreitol and 5% sodium dodecyl sulfate (SDS; vol/vol) separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-HBcAg. For DNA analysis, 20-25 μL of viral samples were treated according to Qiagen QIAamp DNA blood kit protocol. Viral DNA, 2 μL, was amplified by PCR with specific HBV primers. pGEM-1.3xHBV was used for standard calibration. Analysis of HBV DNA replication from cells was performed as described.15

Enzymatic Assay for dNTPs Measurement.

dNTP extraction is based on19 and dNTP level was quantified by DNA polymerase fill-in reaction as described.20

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Generation of Quiescent Tissue Culture Cells by DMSO Induction.

Nondividing cells have minimal amounts of dNTPs that are produced by de novo synthesis. We hypothesized that HBV induces de novo dNTP synthesis in nondividing cells, to ensure sufficient levels of dNTPs for the synthesis of progeny DNA. HBV does not readily infect cells in tissue culture; thus, a commonly used tool for the study of HBV is the hepatic HepG2 cell line stably-tranfected with HBV,21 known as HepG2.2.15, that is active in HBV gene expression and virion production.22 To investigate HBV production in resting cells, we treated HepG2 and HepG2.2.15 cells with DMSO to induce G0/G1 arrest.3, 13 Cells were arrested in a gradual manner and a complete growth arrest was obtained after about 5 days of treatment (Fig. 1A). FACS analysis revealed that both HepG2 and HepG2.2.15 DMSO-treated cells did not incorporate BrdU, indicating that both stopped proliferating (Fig. 1B). Growth arrest was also confirmed by the [3H]thymidine-incorporation assay (Fig. 1C). Finally, in DMSO-treated cells, Ki67 expression, a cell cycle marker, was markedly attenuated over time (Fig. 1D), confirming the quiescent state (G0) of DMSO-treated cells.

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Figure 1. RNR is essential for HBV replication in nondividing cells. (A) DMSO-induced quiescent state in tissue culture cells. HepG2 and HepG2 2.2.15 cells were grown with or without 2% DMSO. Cell density was examined over time by light microscopy (×200). (B) Cells were incubated with 2% DMSO for 6 days and BrdU incorporation was performed after cell wash. After 30 minutes, the cells were fixed and immunostained with fluorescein isothiocyanate (FITC)-BrdU antibody together with PI for FACS analysis. (C) [3H]Thymidine incorporation assay was performed after the cells were washed to measure cell proliferation. (D) Cell cycle marker expression of DMSO-treated cells. Reverse transcription PCR was performed on RNA, which was extracted at different time points after DMSO treatment in HepG2 and HepG2.2.15 cells.

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RNR Activity Is Required for HBV Replication and Viral Production.

HBV replication and virion production was examined in quiescent, DMSO-treated HepG2.2.15 cells. We examined whether RNR, the key enzyme for dNTP synthesis, is required for HBV replication in nondividing cells, by using the specific RNR inhibitor HU.23 Remarkably, the level of HBV replication was dramatically attenuated in HU-treated quiescent HepG2.2.15 cells, as examined by monitoring the intracellular viral DNA in the cytoplasm (Fig. 2A). Next, we quantified the level of secreted virions and revealed that it was higher in the DMSO-treated HepG2.2.15 cells, compared to the nontreated cells (Fig. 2B, lower panel), demonstrating that sufficient levels of dNTPs were available in HepG2.2.15 nondividing cells. In addition, the amount of viral particles released to the medium was sharply reduced as determined by western blot analysis of HBV core protein (Fig. 2C) and PCR-based quantification of viral DNA (Fig. 2B), suggesting that RNR inhibition blocks viral replication and secretion.

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Figure 2. HBV replication is dependent on RNR activity. DMSO-treated HepG2.2.15 cells with or without 1.5 mM HU were analyzed. (A) HBV DNA from cells was isolated, and PCR was performed with HBV-specific primers. HBV plasmid was used as control. (B) HBV DNA isolated from virions was amplified with specific primers. HBV plasmid was used as control, and calibration of viral DNA yield was done using real-time PCR (lower panel). (C) Virions from the cell medium were concentrated by ultracentrifugation and analyzed by SDS-PAGE and immunoblotting with anti-HBcAg.

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HBV Directly Activates R2 Expression and dNTPs Synthesis.

The level of RNR activity is determined by R2 expression, because the R1 protein level is almost constant, while the R2 protein has a short half-life of 3 hours and its gene is not expressed in quiescent cells.10 To examine the effect of HBV on R2 expression, we quantified R2 level in HepG2 and HepG2.2.15 quiescent cells. As expected, the level of R2 transcripts was high in proliferating cells, but undetectable in quiescent DMSO-treated HepG2 cells (Fig. 3A,B). In contrast, R2 was highly expressed in the quiescent DMSO-treated HepG2.2.15 cells. Quiescent cells with no R2 protein generate dNTPs for repair and mitochondrial DNA replication through the low but constitutive expression of p53R2, a cell cycle–independent R2 paralog, which together with R1 forms an active RNR complex.24, 25 The expression level of the p53R2 gene was similar for HepG2 and HepG2.2.15 cells (Fig. 3A), suggesting that HBV affected only R2, and not p53R2, expression. Similar results were obtained at the R2 protein level (Fig. 3C). This suggests that in the presence of HBV, R2 is expressed in quiescent cells.

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Figure 3. R2 is expressed in quiescent HBV-expressing cells. (A,B) Transcript levels of the RNR genes and HBV in proliferative or quiescent (DMSO-treated) cells were detected by (A) reverse transcription PCR (RT-PCR) or (B) real-time PCR. (C) Proteins were extracted from HepG2 and HepG2.2.15 proliferative or quiescent (DMSO-treated) cells and R2 protein level was detected by western blot. (D) Mice were hydrodynamically injected with either control or 1.3xHBV plasmid. After 2 days, RNA was purified from the mice livers and RT-PCR was performed. (E) Primary mouse hepatocytes were isolated and the cells were cultured. After 24 hours, the cells were transfected with different DNA plasmids. After 48 hours, the cells were lysed, RNA was extracted, and RT-PCR was performed. As a negative control for nondividing cells, a proliferative NIH-3T3 cells (mouse cell line) were used, together with the TK1 set of primers for proliferation. Lane 1 = proliferative NIH-3T3, lanes 2-4 = primary mouse hepatocytes transfected with control pCDNA3 (lane 2), wild-type HBV (lane 3), and HBV-XKO (lane 4).

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We next addressed the question whether HBV is directly involved in unscheduled induction of R2 expression in quiescent cells in in vivo system. We employed a 1.3xHBV construct previously used to express HBV in culture and in mice by hydrodynamic injection.15 Expression of the transfected HBV construct in mice livers induced a concomitant increase in the level of R2 (Fig. 3D). This demonstrated that the ability of HBV to induce R2 expression in nondividing cells was not specific to the HepG2.2.15 cell line, and could be reproduced in an independent system. However, liver is not homogenous and may contain some proliferative cells as well. R2 is indeed expressed, although to a low level in the transfected control (Fig. 3D). Therefore, we prepared primary mouse hepatocytes14 to transfect with either 1.3xHBV or 1.3xHBV-XKO, an HBV point mutant which does not express the HBx protein. Remarkably, the wild-type, but not the mutant HBV that is defective in HBx expression, induced R2 transcription (Fig. 3E). Furtheremore, R2 induction was not accompanied by cell proliferation, as examined by the lack of TK1 gene expression. These data suggest that HBV induces R2 expression in quiescent hepatocytes in a HBx-dependent manner.

High Production of dNTPs in Quiescent HBV-Expressing Cells.

Induction of R2 expression should change the intracellular dNTP pools. We quantified deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) concentration using the published protocols.19, 20 The concentration of all the tested dNTPs was reduced by about seven-fold in quiescent HepG2 cells but remained high in HepG2.2.215 cells (Fig. 4A).

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Figure 4. Quiescent HBV-expressing cells produce high amounts of dNTPs. (A) dTTP, dCTP, and dGTP concentration was measured in HepG2 cycling cells and DMSO-treated arrested cells using radioactive polymerase fill-in reaction. (B). HepG2 cells were incubated with conditioned medium from proliferative HepG2 or HepG2.2.15 cells, or from HepG2.2.15 cells treated with DMSO with or without HU (1.5, 2.5, or 5 mM), as indicated. [3H]Thymidine uptake assay was performed.

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According to the metabolic cycles of nucleotides, if synthesis exceeds demands, the deoxynucleotide monophosphate (dNMP) pool increases to a level leading to excretion of deoxyribonucleosides to maintain cell viability.26 Indeed we found that quiescent HepG2.2.15, but not HepG2 cells excreted thymidine at a high level, as was determined by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (Supporting Information Fig. 1). Next, we assayed for thymidine secretion to the medium by determining whether conditioned medium from HepG2.2.15 cells could inhibit [3H]thymidine uptake by competition. Low [3H]thymidine uptake was seen in HepG2 cells that were cultured in conditioned medium taken from either proliferating or nondividing HepG2.2.15 cells (Fig. 4B). To confirm that the excretion of thymidine was due to activation of RNR enzymatic activity, we treated the quiescent HepG2.2.15 cells with HU. The HU-treated cells did not excrete thymidine (Fig. 4B), validating that thymidine excretion is the direct outcome of the high expression and activity of RNR. The process of thymidine excretion is important because both RNR and deoxycytidine monophosphate deaminase, enzymes involved in de novo synthesis, are allosterically inhibited by excess dTTP.27 Thus, excretion of excess thymidine is needed to maintain the activity of the de novo pathway and cell viability.

HBx Viral Protein Is Sufficient to Activate R2 by Interfering with Rfx1 Binding to the R2 Promoter.

To identify the viral protein responsible for the activation of R2, we used lentiviral vectors expressing different HBV constructs (Supporting Information Fig. 2, Supporting Information Methods). The lentiviral system enabled the transduction of these constructs into the nondividing DMSO-treated cells. The infection efficiency was about 100%, as monitored with the lenti–green fluorescent protein (GFP) construct (Supporting Information Fig. 3). As expected, quiescent HepG2 did not express R2 upon infection with lenti-GFP control vector (Fig. 5A). A dramatic 28-fold induction of R2 expression was obtained when the lenti-HBV construct was transduced (Fig. 5A,B). These cells did not proliferate after transduction as measured by cell counts and [3H]thymidine incorporation (Supporting Information Fig. 4), thus demonstrating the surprising fact that R2 gene expression was activated in quiescent cells. HBV encodes the regulatory HBx protein that modulates transcription and other cellular functions (reviewed in Tang et al.28). Remarkably, the expression of HBx alone was sufficient to induce R2 (Fig. 5A,B, lane 3) to a level comparable to that detected in HepG2.2.15 cells. Furtheremore, the HBV construct with a null mutation in the HBx gene12 did not induce R2 expression. These data suggest that HBV induces R2 expression in quiescent cells and that the HBx protein of HBV is required and sufficient in this process.

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Figure 5. HBx induces R2 transcription activation. (A) RNA was extracted and RT-PCR was performed with indicated primers. Lanes 1-4: DMSO-treated HepG2 cells were transduced with lentiviral vector particles and collected for analysis 4 days following transduction. Lane 1 = lenti-GFP, lane 2 = lenti-HBV, lane 3 = lenti-HBx, lane 4 = lenti-HBV-XKO. Lanes 5-7 are controls: lane 5 = DMSO-treated HepG2, lane 6 = DMSO-treated HepG2.2.15, lane 7 = HepG2 proliferating cells. (B) Quantification of R2 messenger RNA (mRNA) level after reverse transcription PCR (RT-PCR) and real-time PCR (N = 5). Experiment and the x-axis labels are as in (A).

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HBx Activates R2 Transcription by Blocking Rfx1 Accessibility to the R2 Promoter.

The R2 gene is under repression by the Rfx1 transcription factor.11 This mechanism of R2 repression was first identified in yeast where the Rfx1 ortholog, Crt1, represses the R2 gene.29 Consistent with this repression role of Rfx1, chromatin immunoprecipitation (ChIP) analysis revealed that Rfx1 was bound to the R2 promoter more effectively in the quiescent cells than in the proliferative cells (Fig. 6A). Remarkably, in the quiescent HBV-expressing HepG2.2.15 cells, Rfx1 did not bind to the R2 promoter, despite the fact that these cells expressed Rfx1 (Supporting Information Fig. 5A). Previously, it has been reported that Rfx1 binds the HBV enhancer to support HBx expression.30, 31 Indeed, ChIP analysis revealed that Rfx1 binds the integrated HBV enhancer in quiescent HepG2.2.15 cells (Supporting Information Fig. 5B). Thus, lack of R2 binding by Rfx1 in HepG2.2.15 cells is specific and might be programmed by HBV. Given the known role of HBx (the HBV regulatory protein) in transcription coactivation, we next asked whether Rfx1 is bound to the R2 promoter in the quiescent HepG2 cells expressing HBx. ChIP analysis on quiescent HepG2 cells transduced with the lentiviral expression vectors revealed that in the presence of HBx, Rfx1 did not bind the R2 promoter (Fig. 6B). Examination of HBx association with the R2 promoter by ChIP analysis of several regions within the R2 promoter (Supporting Information Fig. 6) showed that HBx was associated only with the region that contains the Rfx1 binding site (Fig. 6C). HBx has no reported DNA binding activity, therefore it is likely that HBx is indirectly associated with the R2 promoter, at the binding region of Rfx1, thus preventing Rfx1 access to the R2 promoter. These data suggest that association of HBx with the R2 promoter inhibits Rfx1 binding to the R2 promoter to give rise to R2 transcription activation. Thus, HBx is both required and sufficient to induce R2 expression in quiescent cells.

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Figure 6. HBx induces R2 expression by regulating the association of the transcription repressor Rfx1 with the R2 promoter. (A) ChIP was performed as described in Methods. Briefly, after sonication, an anti-Rfx1 antibody was used for immunoprecipitation (IP). Primers to the R2 promoter were used for PCR amplification. Input (total DNA) controls were taken before immunoprecipitation. (B) ChIP of DMSO-treated HepG2 cells transduced with lentiviral vectors as described in the text. (C) ChIP on cells transfected with pSG5-HA-X was performed as described above with either anti-HA or anti-HBx or control antibody. After DNA extraction, fragments were amplified with primers specific to different regions of the R2 promoter, or to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as indicated.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

HBV generates DNA in the infected cells to form hundreds of genome copies per cell per day. The challenge that the virus faces by infecting nondividing hepatocytes is the limited pool of dNTPs. In large part, the hepatocytes are in a quiescent state and therefore have a pool of dNTPs that cannot support efficient virus production. In the case of HBV, which is replicated via reverse-transcription, activation of the cellular DNA replication machinery is in fact unfavorable, yet the virus needs large dNTP pools. We show here that the virus uses a mechanism enabling it to selectively activate dNTP synthesis by inducing R2 activation without activating the whole cell-cycle program.

In the absence of a reliable system for HBV infection, due to tissue-specificity and species-specificity of the virus, and the fact that hepatoma cell lines are not susceptible to infection, any HBV study is severely hampered. Here, we used a new system in which quiescence-induced tissue culture cells express different HBV constructs upon lenti-HBV infection. In this system, we avoid overexpression effects, which are usually obtained in transfection experiments in proliferating cells. Moreover, our new system of quiescent human hepatocyte tissue culture cells resemble the in vivo HBV infection and enable us to cope with mechanistic viral questions yet to be answered. One of those questions refers to the role of HBx in the HBV life cycle, which has remained a debatable issue. Most of the reported studies were performed in proliferative cultured cells; therefore, the requirement of R2 activation was not evident, a fact that has introduced confusion in the field. We found that HBx, a regulatory protein of HBV, has a critical role for HBV expression in cells. Our data demonstrate that HBx is sufficient to induce R2 expression in quiescent cells, and that HBx expression results in lowered binding of the Rfx1 repressor to the R2 promoter. Given the reports on the role of HBx in cellular DNA repair,32 the possibility of R2 involvement in this process, as well, is of interest and deserves further investigation.

Different viruses have developed diverse strategies to ensure sufficient dNTPs for their life cycle. Retroviruses do not need high levels of dNTPs because they undergo only one cycle of DNA synthesis after infection. The requirement for dNTPs is critical in cases where the viral DNA genome replicates in quiescent cells, as seen in some of the herpesviruses. For example, murine cytomegalovirus (MCMV) replication and DNA synthesis depend on RNR activation in quiescent cells by an as-yet unknown mechanism.33, 34 Lytic DNA viruses have developed different strategies; these viruses increase the cellular dNTP pools by inducing cell proliferation or by degrading the cellular DNA genome or the mitochondrial DNA.35 In this study, we provide evidence that HBV employs a unique mechanism involving activation of R2 transcription to get an adequate amount of dNTPs for its replication in quiescent cells. Furthermore, mammalian cells keep a balanced supply of the four dNTPs as the substrates for DNA replication and repair,24, 27 whereas unbalanced pools can cause genetic abnormalities, high mutation rate, and cell death (reviewed in Reichard27). Also, intracellular nucleotides act as prosurvival factors by binding to cytochrome C and inhibiting the apoptosome.36

Our findings provide an elegant example of a virus manipulation over the host hepatocyte: they not only describe the viral dNTPs synthesis activation mechanism that is crucial for in vivo virulence, but also provide detailed insights into the role of HBx in HBV life-cycle. These observations should contribute to the search for additional antiviral drugs and might have some implications in HBV-related mutagenesis and oncogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. Daniel Tal (Weizmann Institute) for his assistance with the RP-HPLC, and Dr. Hugo Gottlieb (Bar-Ilan University) for the NMR analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_23519_sm_SuppMaterials.doc1135KSupporting Information Materials.

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