Figure 1.

Construction and characterization of 5c3c-derived rHBV expressing IL-21. (A) Schematic representation of plasmid constructs. Open boxes and arrows represent ORFs and promoters, respectively. Stop codons in the S and murine IL-21 (mIL-21) ORFs are indicated by asterisks. Solid diamonds indicate ATG to ACG mutation at preS1 start codon. Solid triangles indicate ATG to ACG mutation at 1^st, 8^th and 11^th codons in human IL-21 (hIL-21) ORF. Restriction enzyme sites (MfeⅠ and MluⅠ) used for inserting cargo sequences and nucleotide positions on HBV genome are indicated. Murine and human IL-21 sequence modifications are detailed in Figure S2 and Figure S3. (B) Supernatants from Huh-7 transfected with indicated plasmids in triplicates were assayed for HBsAg (top) and mIL-21 (bottom) using ELISA. Group means and SEMs are presented. Dotted lines represent cut-off thresholds. (C) Southern blot analysis of intracellular capsid-associated HBV DNA (top), capsid- and virion-associated HBV DNA in the supernatants (middle), and virion-associated DNA in the supernatants captured by anti-preS1 mAb (bottom). Expected presence of HBV capsid and virions in supernatants with or without anti-preS1-capture are indicated below the blots. Transfections were independently repeated with similar results and representative data from one experiment are shown.