Identification of a Hepatitis B Virus S Gene Mutant in Lamivudine-Treated Patients Experiencing HBsAg Seroclearance  Chao-Wei Hsu, Chau-Ting Yeh, Ming-Ling Chang, Yun-Fan Liaw  Gastroenterology  Volume 132, Issue 2, Pages 543-550 (February 2007) DOI: 10.1053/j.gastro.2006.12.001 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Characterization of mutant HBsAg. (A) Northern blot analysis for the expression of wild-type and sP120A mutant. Two plasmids, pCMV-Smt (lanes 2 and 7) and pCMV-S (lanes 3 and 8), encoding the sP120A surface antigen mutant and the wild-type surface antigen were used to transfect Huh-7 cells. The vector pRc/CMV was also used to transfect Huh-7 cells (lanes 1 and 6; as a control). Total RNA extracted from 104 cells was loaded onto an agarose gel (left panel) for Northern blot analysis (right panel). Ten pg of pRc/CMV (lanes 4 and 9) and pCMV-S (lanes 5 and 10) were loaded as negative and positive hybridization controls, respectively. Short lines, 18S and 28S rRNA; arrowhead, mRNA of surface protein. (B) Characterization of the wild-type and mutant HBsAg by SDS-PAGE. Huh-7 cells were transfected with pRc/CMV (lane 1, Mock), pCMV-S (lane 2, WT), and pCMV-Smt (lane 3, P120A), respectively. The culture medium (upper panel) as well as the cytoplasmic fraction (middle panel) was collected for SDS-PAGE analysis. Surface proteins were detected with a rabbit polyclonal anti-HBs antibody (upper and middle panels). β-actin was also detected as a control (lower panel). Short lines, the positions of GP27, P24, and actin; triangle, a nonspecific background band. (C) Immunofluorescence analysis of the wild-type and mutant HBsAg. Huh-7 cells were transfected with pCMV-Smt (sP120A, upper 2 rows) and pCMV-S (wild type, lower 2 rows) to express the mutant and wild-type HBsAg, respectively. HBsAg was detected by use of the rabbit polyclonal anti-HBs antibody as the primary antibody (left column). DAPI staining was performed to visualize the nuclei (middle column). Pictures in these 2 columns were merged to demonstrate the subcellular localization of HBsAg (right column). (D) Detection of secreted wild-type and mutant HBsAg by monoclonal antibodies. Two hundred microliters of culture medium containing the mutant (mt) and wild-type (wt) surface protein were loaded onto a nitrocellulose membrane for Western analysis. Four different monoclonal antibodies, MAHBs 1–4 (MO1–4) as well as a polyclonal (PO) antibody, were used for detection (see Materials and Methods section). Gastroenterology 2007 132, 543-550DOI: (10.1053/j.gastro.2006.12.001) Copyright © 2007 AGA Institute Terms and Conditions