Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts

Lembo, David; Gribaudo, Giorgio; Hofer, Anders; Riera, Ludovica; Cornaglia, Maura; Mondo, Alessandra; Angeretti, A; Gariglio, Marisa; Thelander, Lars; Landolfo, Santo

doi:10.1128/jvi.74.24.11557-11565.2000

Cited by 40 publications

(55 citation statements)

References 68 publications

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“…For example, murine cytomegalovirus (MCMV) replication and DNA synthesis depend on RNR activation in quiescent cells by an asyet 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.…”

Section: Discussionmentioning

confidence: 99%

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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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 10 7 to 10 11 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;51:1538-1546 H epatitis 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 doublestranded 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 lM. 4 The Michaelis constant (K m ) of the viral polymerase at a dNTP concentration of 0.4 lM is only 62%, whereas a 4 lM 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 Â 10 5 to 2 Â 10 6 dNTPs are consumed per day in this process. Considering cell volume as 500 fL, 7 a resting cell contains approximately 1.2 Â 10 5 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 cycl...

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Section: Discussionmentioning

confidence: 99%

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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“…Transfer of proteins to a Hybond-C Extra membrane was made as previously described (11). After transfer, the membrane was blocked in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween 20 containing 2% dried milk for 30 min before incubation with either a polyclonal rabbit antip53R2 antibody (ab2014, Abcam), a polyclonal rabbit anti-R2 antibody (33), or a monoclonal mouse anti-R1 antibody, AD203 (13), for 1 h at room temperature or overnight at 4°C. A minor cross-reaction was found between the anti-p53R2 and anti-R2 antibodies, but this was not a problem since these two proteins have different electrophoretic mobilities due to their different sizes (5).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Mammalian Ribonucleotide Reduction and dNTP Pools after DNA Damage and in Resting Cells

Håkansson

Hofer

Thelander

2006

Journal of Biological Chemistry

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224

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Ribonucleotide reductase (RNR) provides the cell with a balanced supply of deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. In budding yeast DNA damage leads to an up-regulation of RNR activity and an increase in dNTP pools, which are essential for survival. Mammalian cells contain three non-identical subunits of RNR; that is, one homodimeric large subunit, R1, carrying the catalytic site and two variants of the homodimeric small subunit, R2 and the p53-inducible p53R2, each containing a tyrosyl free radical essential for catalysis. S-phase-specific DNA replication is supported by an RNR consisting of the R1 and R2 subunits. In contrast, DNA damage induces expression of the R1 and the p53R2 subunits. We now show that neither logarithmically growing nor G o /G 1 -synchronized mammalian cells show any major increase in their dNTP pools after DNA damage. However, non-dividing fibroblasts expressing the p53R2 protein, but not the R2 protein, have reduced dNTP levels if exposed to the RNR-specific inhibitor hydroxyurea, strongly indicating that there is ribonucleotide reduction in resting cells. The slow, 4-fold increase in p53R2 protein expression after DNA damage results in a less than 2-fold increase in the dNTP pools in G o /G 1 cells, where the pools are about 5% that of the size of the pools in S-phase cells. Our results emphasize the importance of the low constitutive levels of p53R2 in mammalian cells, which together with low levels of R1 protein may be essential for the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis in G o /G 1 cells. Mammalian cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs)2 for DNA replication and repair. The rate-limiting step in the formation of DNA precursors is the de novo reduction of ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1). In S phase, the mammalian RNR enzyme is composed of the homodimeric R1 and R2 subunits, which together form a heterotetrameric active enzyme. The large R1 protein (90 kDa) carries the active site, whereas the small R2 protein (45 kDa) contains a diferric iron center generating a tyrosyl free radical necessary for catalysis (1, 2). An additional mammalian RNR protein, p53R2, was identified in 2000 (3, 4). Like the homologous R2 protein, p53R2 contains a tyrosyl free radical and forms an active RNR complex with the R1 protein in vitro (5). The tyrosyl free radical of both the R2 and the p53R2 proteins is specifically destroyed by the RNR inhibitor hydroxyurea (5, 6).Because unbalanced dNTP pools can cause genetic abnormalities and cell death (1), RNR activity is tightly regulated in mammalian cells by S-phase-specific transcription of the R1 and R2 genes (7-9), binding of nucleoside triphosphate allosteric effectors to the R1 protein (10), and anaphase promoting complex-Cdh1-mediated degradation of the R2 protein in late mitosis (11,12). In cycling cells, the S-phase-dependent activity of the RNR complex is l...

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“…Splenocytes from B16FL-injected mice were gamma-irradiated and pulsed with peptide at 10 Ϫ8 M, and cultured with splenocytes from MCMV-infected mice in RPMI 1640 supplemented with 10% FBS for 3 days, after which 10 U/ml rIL-2 (eBioscience) was added. After 10 days, the percentage of CD8 T cells responding to the simulating peptide epitopes was assessed by intracellular cytokine staining, and the cells were used in 51 Cr release assays.…”

Section: T Cell Linesmentioning

confidence: 99%

Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis

Pinto

Munks

Koszinowski

et al. 2006

The Journal of Immunology

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Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2b-restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2b-restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on Kb vs Db, but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV’s three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells.

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Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts

Cited by 40 publications

References 68 publications

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Regulation of Mammalian Ribonucleotide Reduction and dNTP Pools after DNA Damage and in Resting Cells

Coordinated Function of Murine Cytomegalovirus Genes Completely Inhibits CTL Lysis

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