HBV cccDNA Southern blot was conducted following a similar procedure as described by Summers et al. (1990) with modifications. Briefly, to selectively extract HBV cccDNA, infected hepG2-NTCP cells in 6-cm dishes were lysed with 1 ml lysis buffer at 37°C for 60 min, followed by addition of 0.25 ml of 2.5 M KCl and incubation at 4°C overnight. The lysis buffer was not supplemented with proteinase K, containing 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1% SDS. The lysate was then clarified by centrifugation at 12,000 g for 30 min at 4°C and extracted with phenol and phenol:chloroform. DNA was precipitated with equal volume of isopropanol in the presence of 20 µg glycogen (Roche) and finally dissolved in TE buffer. The prepared DNA sample was then treated with plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease DNase (Epicentre Technologies) following manufacturer's instructions. For Southern blotting, the plasmid-safe DNase-treated DNA was separated on a 1.3% agarose gel and then transferred to a nylon membrane (Hybond-N + ; Amersham) using a standard neutral transfer procedure. A 3280-bp plasmid constructed by inserting a 588-bp HBV DNA fragment (from 1805 to 2392, genotype D, Southern blot probe) into a 2692 bp pMD18T vector (Takara) was also run on the same agarose gel to serve as the molecular marker for cccDNA in Southern blot analysis. The plasmid is of similar size of HBV genome and was mainly in supercoiled form; therefore it runs at similar size as HBV cccDNA in agarose gel. The nylon membrane was hybridized with a [α-32 P] dCTP-labeled HBV probe (genotype D HBV DNA fragment from 1805 to 2392) prepared by random primer DNA labeling kit (Ver.2.0; Takara). Hybridization was carried out in 7 ml of Perfect Hyb Plus Hybridization Buffer (Sigma) with 1 hr pre-hybridization, followed by overnight hybridization at 67°C. The membrane was then washed once with 2× SSC/0.1% SDS, 1× SSC/0.1% SDS, and 0.5× SSC/0.1% SDS at 67°C for 20 min, respectively. Finally, the membrane was subjected to autoradiographic exposure.