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. 2019 Apr 26;15(4):e1007742. doi: 10.1371/journal.ppat.1007742

Fig 7. Pol α is required for the formation of covalently closed minus strand rcDNA.

Fig 7

(A) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal. From day 6, PFA was removed from culture media for 24 h in the presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with DMSO as a control. Cytoplasmic HBV core DNA was extracted and analyzed by Southern blot hybridization by using HBV (-) probe to hybridize with viral plus strand. (B) HepAD38 cells were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. At day 4, the cells were re-seeded and transfected with 10 pmol siRNA targeting POLΑ1 or scramble siRNA, by using RNAiMAX. From 48 h to 72 h after siRNA transfection (day 6 to day 7 after tet removal), PFA was removed to resume viral DNA synthesis. Cytoplasmic HBV core DNA was extracted and analyzed by Southern blot hybridization by using HBV (-) probe to hybridize with viral plus strand DNA. (C) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal, HBV intracellular nucleocapsids were purified by 30% sucrose cushion ultracentrifugation. In vitro EPR assay was performed at 37°C for 16 h in the absence or presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with or without 0.1 mM of each dNTP. Upon completion of EPR reaction, viral DNA was extracted and analyzed by Southern blot hybridization. (D) HepAD38 cells were cultured in the absence of tet and HBV DNA replication was arrested by PFA treatment between day 2 to 6 after tet removal. The cells were then cultured in the presence of tet and absence of PFA for 24 h. Hirt DNA without prior treatment, treated with Exo I & III without or with following EcoRI digestion were resolved by agarose gel electrophoresis and detected by Southern blot hybridization with a minus-strand specific probe. (E) HepAD38 cells were cultured in the presence of PFA from day 2 to day 6 after tet removal. From day 6, cccDNA synthesis was initiated by removing PFA for 24 h in the presence of Pol α inhibitor APH (1 μM) or CD437 (1 μM), with DMSO as a control. Hirt DNA was extracted and analyzed by Southern blot hybridization without prior heat denaturation, or after Exo I & III digestion. Minus strand covalently closed DNA is denoted.