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

Abstract

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...

Hepatitis B virus induces RNR-R2 expression via DNA damage response activation

“…RNR-dependent dNTP production is crucial for HBV propagation, and its inhibition by hydroxyurea (HU) halts mature HBV virion production [7]. Remarkably, HBV induces RRM2 expression in quiescent cells [7], [8].…”

Modulation of Cell Proliferation Pathways by the Hepatitis B Virus X Protein: A Potential Contributor to the Development of Hepatocellular Carcinoma

“…Finally, the upregulation of the R2 subunit of ribonucleotide reductase by HBx, and the dependence of HBV replication on upregulation of R2 expression suggest that HBx may cause cell cycle entry in order to increase the availability of dNTPs. Recently, it was shown that HBV can increase the cellular concentration of dNTPs in HepG2.2.15 cells, as compared to HepG2 cells (Cohen et al, 2010). Although these findings are intriguing, this increased pool of dNTPs was not directly linked to HBx and not confirmed in HBV-infected normal hepatocytes.…”

Hepatitis B Virus X Protein: A Key Regulator of the Virus Life Cycle

“…While this effect of HBx on cell cycle progression can probably lead to carcinogenesis and thus become deleterious for the host, it is believed that it might be important for the virus to induce expansion of available deoxynucleoside triphosphate pools within the cells which it needs for replication (Bouchard et al, 2003). Indeed, using HepG2 cells, it was reported that HBx is sufficient for the induction of the R2 subunit of the ribonucleotide reductase (RNR) (Cohen et al, 2010). RNR is the key enzyme responsible for de novo dNTP synthesis and is composed of R1 and R2 subunits (Nordlund & Reichard, 2006).…”

“…While the R1 subunit is expressed in quiescent cells, the R2 subunit expression is silenced (Chabes et al, 2003). As a consequence of induction of R2 by HBx, the dNTP pool for effective viral production was increased without affecting cell cycle progression (Cohen et al, 2010). Different groups using different models showed that HBx may localize and interact with the proteasome components thereby influencing proteasome subunit composition (Chen et al, 2001;Fischer et al, 1995;Hu et al, 1999;Zhang et al, 2000).…”