RESUMO
The small (S) envelope protein is the major component of hepatitis B virus surface antigen (HBsAg). Some mutations in the S-HBsAg coding region may cause deficiency in the secretion of both viral and empty subviral particles (SVPs) and lead to accumulation of HBsAg inside the cells. In this study, we identified a unique amino acid substitution (L215Q) in the carboxyl-terminal end of S-HBsAg of an HBV genotype F isolate that provoked an inhibitory effect on secretion of SVPs. HBsAg levels were measured daily by enzyme-linked immunosorbent assay in the medium and cell extracts of HuH7 and CHO cells transiently transfected with plasmids containing wild-type or mutated S-HBsAg gene. Wild-type HBsAg was detectable in both medium and cell extracts of transfected cells. In contrast, extracellular levels of mutant HBsAg were not higher than cut-off values. By immunofluorescence with monoclonal antibody, it was shown that wild-type HBsAg was distributed throughout the cytoplasm, whereas mutant HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. Amino acid substitutions that inhibit HBsAg secretion, such as that characterized in this study (L215Q), should have implications in HBV immunological diagnostics.
Assuntos
Substituição de Aminoácidos , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírion/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Citoplasma/metabolismo , Genótipo , Vírus da Hepatite B/classificação , Humanos , Microscopia de Fluorescência , Dados de Sequência MolecularRESUMO
The impact of hepatitis B virus (HBV) genotypes on the sensitivity of surface antigen (HBsAg) detection assays has been poorly investigated. Here, plasmids carrying consensus or variant coding sequences for HBV surface proteins from genotypes A, D and F, were constructed. HBsAg levels were evaluated in medium and extracts of transfected CHO cells by a commercial polyclonal-based assay. We show that HBsAg detection values of consensus forms from genotypes D and F were, respectively, 37% and 30% lower than those obtained by genotype A. However, the presence of two single variations, T143M in genotype A, and T125M in genotype D, produced a decrease of 44% and an increase of 34%, respectively, on HBsAg mean values in comparison with their consensus forms. In conclusion, HBsAg detection levels varied among HBV genotypes. However, unique amino acid substitutions not linked to genotypes, such as T125M and T143M described here, should have more implications in HBV immunological diagnostics than the set of variations characteristic of each HBV genotype.
Assuntos
DNA Viral/análise , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B/virologia , Sequência de Aminoácidos , Animais , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Genótipo , Dados de Sequência MolecularRESUMO
The impact of hepatitis B virus (HBV) genotypes on the sensitivity of surface antigen (HBsAg) detection assays has been poorly investigated. Here, plasmids carrying consensus or variant coding sequences for HBV surface proteins from genotypes A, D and F, were constructed. HBsAg levels were evaluated in medium and extracts of transfected CHO cells by a commercial polyclonal-based assay. We show that HBsAg detection values of consensus forms from genotypes D and F were, respectively, 37 percent and 30 percent lower than those obtained by genotype A. However, the presence of two single variations, T143M in genotype A, and T125M in genotype D, produced a decrease of 44 percent and an increase of 34 percent, respectively, on HBsAg mean values in comparison with their consensus forms. In conclusion, HBsAg detection levels varied among HBV genotypes. However, unique amino acid substitutions not linked to genotypes, such as T125M and T143M described here, should have more implications in HBV immunological diagnostics than the set of variations characteristic of each HBV genotype.