D S Kostyushev1, A P Zueva2, S A Brezgin3, A D Lipatnikov4, V N Simirskii5, D Glebe6, E V Volchkova7, G A Shipulin1, V P Chulanov3. 1. Central Research Institute of Epidemiology, Moscow, Russia. 2. Central Research Institute of Epidemiology, Moscow, Russia; M.V. Lomonosov Moscow State University, Moscow, Russia. 3. Central Research Institute of Epidemiology, Moscow, Russia; I.M. Sechenov First State Medical University, Moscow, Russia. 4. Central Research Institute of Epidemiology, Moscow, Russia; D.I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia. 5. N.K. Koltzov Institute of Developmental Biology, Moscow, Russia. 6. Justus-Liebig University of Giessen, Institute of Medical Virology, Giessen, Germany. 7. I.M. Sechenov First State Medical University, Moscow, Russia.
Abstract
AIM: To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. MATERIAL AND METHODS: Human hepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. RESULTS: DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P<0.005) after transfection of DNMT3A, but is unaltered under DNMT1 treatment. CONCLUSION: DNMT3A regulates the size of cccDNA pool and is important for persistency of HBV infection.
AIM: To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. MATERIAL AND METHODS:Humanhepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. RESULTS:DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P<0.005) after transfection of DNMT3A, but is unaltered under DNMT1 treatment. CONCLUSION:DNMT3A regulates the size of cccDNA pool and is important for persistency of HBV infection.
Entities:
Keywords:
DNA-methyltransferases (DNMT); PCR; cccDNA; chronic hepatitis B (CHB); hepatitis B virus (HBV); pgRNA