Molecular Cloning of HBV and AMPK Constructs

Wild type (WT) and HBx-deficient mutant constructs of replication-competent 1.3 mer HBV subtype ayw were kindly provided by Dr. Wang-Shick Ryu (Yonsei Univ, South Korea). pCDH-hNTCP-C9 generated from the human NTCP-C9 (hNTCP-C9) construct in pcDNA6.1, kindly provided by W. Li [30 (link)], has been described [31 (link)]. The codon optimized CaMKII α gene was synthesized and inserted into an EcoR I-linearized pBHA plasmid, resulting in the plasmid pBHA-CaMKII α. To generate pCMV10-HA-CaMKII α, PCR-amplified CaMKII α DNA (Table S1) from pBHA-CaMKII α was subcloned into the EcoR I-linearized vector pCMV10-HA. To generate pCMV10-Myc-AMPK α1, PCR-amplified AMPK α1 DNA from pHA-AMPK α1 (provided by Dr. Joohun Ha) [32 (link)] (Table S1) was subcloned into the Msc I- and EcoR I-linearized vector pCMV10-Myc. A lentiviral expression vector encoding the HA-CaMKII α gene was generated by inserting Xba I- and BamH I-digested HA-CaMKII α DNA into the linearized pCDH-CMV-MCS-EF1-Puro (System Biosciences; CD510B-1) vector, and a lentiviral encoding the Myc-AMPK α1 gene was generated by inserting Xba I- and EcoR I-digested Myc-AMPK α1 DNA into linearized pCDH-CMV-MCS-EF1-Puro. The luciferase reporter pGL3-EnhII/Cp, pGL3-EnhI/Xp, pGL3-preS1p, and pGL3-preS2p constructs have been described previously [33 (link)].