Journal: PLoS Pathogens
Article Title: Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis
doi: 10.1371/journal.ppat.1010057
Figure Lengend Snippet: (A) Illustration of wild-type precore protein (p25-WT) and mutant/HA-tagged p25 as well as its proteolytically processed products p22 and p17 that are expressed by pXF3H-derived plasmids (see for detailed description). Red arrow indicates the mutation of Cp starting codon (AUG) to AUA. The gray box indicates the insertion of HA tag at the immediate upstream of Cp starting codon. (B) HepG2 cells were transfected with pXF3H-p25-WT and derived plasmid expressing p25 with the indicated single amino acid substitution. The cells were harvested at 48 h post transfection. Intracellular p25-derived proteins were detected by Western blot assay with an antibody recognizing the last 14 amino acid residues at the C-terminus of precore protein and Cp (anti-HBc-170A). HBeAg in culture media was detected by CLIA and results (mean ± SD) from three independent experiments were analyzed by two-tailed Student’s t-test (unpaired), ***: P < 0.001. (C) HepG2 cells were transfected with a pXF3H-derived plasmid expressing the indicated p25 proteins or pCMV-HBc expressing Cp. Intracellular p25-derived proteins or Cp were detected by Western blot assay with an anti-HBc-170A as the primary antibody. The lysate from AML12HBVpolY63F cells was analyzed in parallel to serve as the control of hyper- and hypo-phosphorylated Cp. (D) HepG2 cells were transfected with pXF3H-p25HA, harvested at 48 h post transfection and lysed with core DNA lysis buffer. Aliquots of the cell lysate were mock-treated or treated with Lambda phosphatase at 30°C for 40 min. HBV p22 in the reactions were detected by Western blot assay with anti-HBc-170A. ( E ) HepG2 cells were transfected with a plasmid expressing the indicated precore protein and harvested at 48 h post transfection. The cells were lysed by 1× LDS lysis buffer (contained 2.5% B-ME) and the lysates were resolved by electrophoresis in NuPAGE 12% Bis-Tris Protein Gel (upper panel) and 12% phos-tag gel (lower panel). After blotting onto membranes, p22 were detected by anti-HBc-170A as primary antibody. β-actin served as a loading control.
Article Snippet: HepG2 cells transfected with desired plasmids were lysed by core DNA lysis buffer, 40 μl lysed samples were incubated with 1μl Lambda phosphatase (NEB, MA, USA), 5 μl 10 ×NEB buffer 3.1 and 4 μl nuclease-free water for 40 min at 30°C.
Techniques: Mutagenesis, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Two Tailed Test, Lysis, Electrophoresis
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