Figure 1.

Up-regulation of apoptosis inhibitors induced by hepatitis B virus (HBV) replication in primary human hepatocyte (PHHs). (A) Formation of HBV covalently closed circular DNA (cccDNA) in HBV-infected PHHs was examined by real-time polymerase chain reaction (PCR) using total DNA extracted from mock- (control) or HBV-infected PHH cultures on day 4 postinjection (p.i.). DNA extracted from high-titer (>5 × 10e8/mL) serum of an HBV-positive patient and from serum of an HBV-positive chimpanzee containing HBV relaxed circular DNA were used as specificity (negative) controls. Total liver DNA extracted from HBV-positive patient and plasmid DNA encoding for HBV 1.3-overlength genome served as positive controls. PCR products were separated in a 2% agarose gel, water control, and DNA size marker (M) are indicated. (B) Hepatitis B early antigen (HBeAg) was measures in cell culture supernatants from day 1 to 4 p.i. of HBV-infected PHHs prepared from 2 different donors (PHH4 and PHH5) as signal-to-control ratio. Day 1 values reflect input virus. Values are shown as mean ± SD (n = 3; ***P < .001; Student's t test). (C) PHH cultures were infected with AdG-HBV1.3 at a multiplicity of infection of 1–2 infectious units per cell. Expression of green fluorescent protein was visualized by fluorescence microscopy at 24 hours p.i. (D) Expression of cIAP2, Mn-SOD, and β-actin (loading control) in mock- or HBV-infected PHH4 and PHH5 was analyzed by Western blot analysis of total protein extracts prepared on day 4 p.i. Levels of interferon regulatory factor 3 (IRF3) and lamin (loading control) in the nucleus of mock- or HBV-infected PHH4 and PHH5 were determined by Western blot analysis of nuclear proteins extracted on day 4 p.i. (left panel). Densitometric quantification (right panel) of cIAP2 or Mn-SOD relative to β-actin and interferon regulatory factor 3 (IRF3) relative to lamin was performed using ImageJ software. Protein levels are given relative to respective mock control (set to 1).