Figure 2.
Cesium chloride (CsCl) density equilibrated centrifugation of wild‐type hepatitis B virus (HBV) and recombinant HBV expressing NanoLuc (HBV/NL). Supernatants of HepG2 cells transfected with pUC1.2xHBV (a), pUC1.2xHBV/NL plus pUC1.2xHBV‐D (b), and pUC1.2xHBV‐D (c) were centrifuged at 150 000 g for 50 h in a CsCl gradient. An aliquot from the top of the centrifuged tube was collected. Hepatitis B surface antigen (HBsAg; blue) in 10% of each aliquot was quantified by automated ELISA. The amount of NL DNA (dotted blue line) (b) and HBV DNA (dotted black line) (a, c) in 10% of each fraction was measured by quantitative PCR using a primer set for the NL gene and HBV DNA, respectively. Infectivity of 25% of each fraction in a and b was assayed by infecting 2 × 105 HepG2/NTCP cells and measuring HBV DNA by quantitative PCR for wild HBV (green) (a) and NL activity for HBV/NL (red) (b) of 10% of the total cell lysate. Pink dots denote buoyant density. Southern blot analysis of each fraction was carried out by probing with digoxigenin‐labeled HBV DNA.