Scrambled sc shrna

The plasmid pCMV-3 × HA1-HBx (HA-HBx) encodes full-length HBx (genotype D) downstream of the three copies of the influenza virus hemagglutinin (HA) [27 (link)]. The 1.2-mer WT HBV replicon containing 1.2 units of the HBV genome (genotype D) and its HBx-null counterpart have been described previously [28 (link)]. The plasmids pCMVT N-HA-hE6AP encoding human HA-tagged E6AP (amino acids 262–853) and pCH110 encoding the Escherichia coli β-galactosidase (β-Gal) gene were purchased from Addgene (Watertown, MA, USA). The plasmid RC210241, encoding the human Na⁺-taurocholate cotransporting polypeptide (NTCP), was obtained from OriGene (Rockville, MD, USA). Scrambled (SC) shRNA, E6AP shRNA, and p53 shRNA plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For the mammalian two-hybrid assay, the PCR fragment from pCMVT N-HA-hE6AP encoding E6AP (amino acids 262–853) was cloned in pSG424, in-frame and downstream of Gal4 (amino acids 1–147) [29 (link)] to generate G4-E6AP. In addition, the PCR fragment encoding HBx (amino acids 1–154) from pCMV-3 × HA1-HBx was fused upstream of the VP16 activation domain (amino acids 423–490) in pCMV-VP16 [30 (link)] to generate HBx-VP16. The reporter G5E1b-luc contained five copies of the GAL4 binding site upstream of a minimal E1b promoter in pGL3 (Promega, Madison, WI, USA), as described previously [30 (link)].

The plasmid pCMV-3 × HA1-Core [32 (link)] contains the complete HCV Core (genotype 1b) sequence positioned downstream of three copies of the influenza virus hemagglutinin (HA) epitope. p53 small hairpin RNA (shRNA), scrambled (SC) shRNA, and E6AP shRNA plasmids were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP, was purchased from Addgene (Watertown, MA, USA). The pCMV p53-WT and pHA-ubiquitin (Ub) plasmids were generously provided by Dr. C.-W. Lee (Sungkyunkwan University, Korea) and Dr. Y. Xiong (University of North Carolina at Chapel Hill, NC, USA), respectively.