Figure 7.

5dCG-delivered RNA interference inhibited co-infecting wild type HBV. (A) Southern blot analysis of replication of 5dCG constructs harboring HBV-targeting siRNA precursors. Intracellular capsid-associated HBV DNA was prepared from Huh-7 cells transfected with indicated plasmids. Ci and Xi denote expression cassettes of siRNA precursor targeting C and X ORF, respectively. Cm and Xm denote synonymous mutations in C and X ORF that confer resistance to Ci and Xi, respectively. Transfection efficiency was normalized using relative luciferase activities expressed by co-transfected firefly luciferase reporter measured using Luciferase Assay System (Promega); (B) ELISA analysis of HBsAg in culture media of infected PTH. Freshly prepared PTH were infected with wild type HBV and/or transfected Huh-7 cell supernatants as indicated. Media were changed and collected at indicated time points, and assayed for HBsAg; (C) Immunofluorescent analysis of intracellular HBcAg expression in infected PTH. Infected cells were fixed at 15 days post infection and intracellular HBcAg was detected using anti-HBcAg (Dako). Percentages of HBcAg-positive cells were calculated from ten randomly selected views and listed on the right. Cell nuclei were stained using DAPI. Scale bars, 200 µm.