PMC

Immunofluorescence staining of surface proteins in wild-type genome and pre-S deletion mutants -transfected Huh-7 cells. Huh-7 cells were first seeded on the chamber slide and incubated at 37°C overnight. After transient transfection of wild-type and pre-S deletion mutants, the slides were stained with monoclonal antibodies against pre-S1, anti-pre-S1 and S, anti-HBs. The endoplasmic reticulum (ER) of the cells was double-immunostained with anti-calnexin antibody. The nuclei of the cells were counterstained with Hoechst. Images were examined under an Olympus fluorescent microscope (original magnifications x200).

Analysis of HBV RNA transcripts. HBV RNA was analyzed three days after transfection of Huh7 cells with wild-type genome or variant HBV DNAs. NC, negative control, only pGEM-4z transfected. (A) Exemplary Northern blot of total RNA detected with a full-length HBV DNA probe. GAPDH RNA was detected as loading control. (B) Quantitative evaluation of the 3.5-kb pg (pregenomic)/preC (precore)-RNA signals in Northern blots relative to wild-type was analyzed by Image J software. Mean values and standard errors of at least three independent experiments are indicated. (C) Quantitative evaluation of preS1-mRNA and pre-S2/S-mRNA signals relative to wild-type was performed by Image J software. Mean values and standard errors of at least three independent experiments are shown. * indicates p < 0.05; ** indicates p < 0.01.

Analysis of secreted and intracellular HBV proteins three days after transfection of Huh7 cells with wild-type and pre-S deletion variant DNAs. NC, negative control, only vector pGEM-4z transfected. (A) HBsAg and HBeAg levels in cell culture supernatant (diluted 1:10) relative to wild-type levels detected by ELISA. Mean values and standard errors of at least four experiments are shown. * indicates p < 0.05; ** indicates p < 0.01. (B) Western blots of cell lysate and cell culture supernatant were used to detect HBV surface proteins with primary antibodies anti-pre-S1 and anti-HBs; HBV core and HBeAg were detected by rabbit polyclonal antibody anti-HBV core. Detection was carried out with peroxidase-coupled secondary antibodies and chemiluminescence substrate. Detection with anti-actin antibody was performed as loading control. The white triangles, gray triangles and asterisks indicate large, middle and small surface protein, respectively. (C) Quantitative evaluation of intracellular and extracellular viral proteins relative to wild-type was analyzed by Image J software. Mean values and standard errors of at least three independent experiments are indicated. * indicates p < 0.05; ** indicates p < 0.01.

Map of HBV pre-S region and viral genomes. (A) Functional domains within the HBV pre-S region. The pre-S region consists of the pre-S1 and pre-S2 domains. The pre-S1 domain contains 119 amino acids (used in this study) and is further divided into two parts, N half (aa 1 to 57) and C half (aa 58 to 119). The pre-S2 domain contains 55 amino acids. The pre-S domain contains multiple functions as shown. N-half of pre-S1 contains hepatocyte binding site essential for infection. C-half of pre-S1 contains a site important for dual topology of L proteins and a nucleocapsid binding site for virion morphogenesis. C-half of pre-S1 also contains S-promoter necessary for expression of S gene. Pre-S2 domain has pHSA (polymerized human serum albumin) binging site. Black triangle, myristylation at second amino acid; white triangle, N-link glycosylation at N-4 of the M protein; gray triangle, O-link glycosylation at T-37 of the M protein. (B) Plasmids encoding the wild-type (wild-type1.2) and pre-S deletion HBV genome. HBV sequence is shown by heavy line and flanking plasmid pGEM-4z sequence shown by the thin line. ORFs for pre-C (pC), C, P, pre-S1 (pS1), pre-S2 (pS2), S, and × genes are drawn as boxes. Arrows above the ORF boxes show the start sites for the pregenomic/Core (3.5 kb), pre-S1 (2.4 kb), pre-S2/S (2.1 kb), and × (0.8 kb) mRNA. Relevant endonuclease restriction sites and positions are indicated. Map of wild-type pre-S1/S2 domain is shown and the number above the map indicates the amino acid site of the defined domain. Gray box indicates the deleted region and the number in the box indicates the length of this box. The deletion of five functional sites are indicated and shown at right hand column. S, T, N, M, and P indicate 5 functional sites, S-promoter, topology, nucleocapsid binding site, the start codon of M protein, and pHSA site respectively. + and - indicate presence of deletion and absence of deletion respectively.