title: Hepatitis B virus induces RNR-R2 expression via DNA damage response activation.
abstract: OBJECTIVE
  Hepatitis B virus (HBV) infects and replicates in quiescent hepatocytes, which are deficient in dNTPs, the critical precursors of HBV replication. Most tumor viruses promote dNTP production in host cells by inducing cell proliferation. Although HBV is known as a major cause of hepatocellular carcinoma, it does not lead to cellular proliferation. Instead, HBV acquires dNTPs by activating the expression of the R2 subunit of the Ribonucleotide Reductase (RNR) holoenzyme, the cell cycle gene that is rate-limiting for generation of dNTPs, without inducing the cell cycle. We wished to elucidate the molecular basis of HBV-dependent R2 expression in quiescent cells.
  
  
  METHODS
  Quiescent HepG2 cells were transduced with an HBV-containing lentiviral vector, and primary human hepatocytes were infected with HBV. DNA damage response and RNR-R2 gene expression were monitored under this condition.
  
  
  RESULTS
  We report here that HBV-induced R2 expression is mediated by the E2F1 transcription factor, and that HBV induces E2F1 accumulation, modification and binding to the R2 promoter. We found that Chk1, a known E2F1 kinase that functions in response to DNA damage, was activated by HBV. In cells where Chk1 was pharmacologically inhibited, or depleted by shRNA-mediated knockdown, HBV-mediated R2 expression was severely attenuated. Furthermore, we found that HBV attenuates DNA repair, thus reducing cellular dNTP consumption.
  
  
  CONCLUSIONS
  Our findings demonstrate that HBV exploits the Chk1-E2F1 axis of the DNA damage response pathway to induce R2 expression in a cell cycle-independent manner. This suggests that inhibition of this pathway may have a therapeutic value for HBV carriers.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Ricardo-Lax
authors.affiliation: Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Vyas
authors.initials: V
authors.lastname: Ramanan
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Shamia
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Nina
authors.initials: N
authors.lastname: Reuven
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States. Electronic address: amirsh9@clalit.org.il.
authors.forename: Amir
authors.initials: A
authors.lastname: Shlomai
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address: yosef.shaul@weizmann.ac.il.
authors.forename: Yosef
authors.initials: Y
authors.lastname: Shaul
mesh_terms: D017209:Apoptosis; D015139:Blotting, Southern; D015153:Blotting, Western; D002453:Cell Cycle; D002455:Cell Division; D049109:Cell Proliferation; D004249:DNA Damage / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D015967:Gene Expression Regulation, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006526:Hepatitis C / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics*; D014775:Virus Activation / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit
keywords: Chk1; DDR; E2F1; HepG2; Primary human hepatocytes; Quiescent cells; RRM2; dNTPs