Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies.
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Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection. (+info)
Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.
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The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA. (+info)
Hepatitis delta virus genome replication: a polyadenylated mRNA for delta antigen.
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Hepatitis delta virus (HDV) replicates its genome in the nucleus of an infected cell. However, an unsolved problem has been the identification in the cytoplasm of a putative mRNA for the synthesis of the only virus-coded protein, the delta antigen. We now report the characterization of an 800-base RNA that is cytoplasmic, polyadenylated, and antigenomic and that should direct the translation of the delta antigen. This RNA was about 500 times less abundant than full-length genomic RNA. We mapped the predominant 5' terminus and also the 3' site at which the poly(A) is added. At a point 15 to 20 bases upstream of the poly(A) addition site is the sequence AAUAAA, which could have been used as a signal for the polyadenylation. When an infectious cDNA clone of the whole HDV genome was changed at this site to UUUAAA, the clone was no longer infectious and it was unable to direct the synthesis of the delta antigen. These findings provided additional evidence that the polyadenylated RNA was at least the predominant method for the expression of the delta antigen. Apparently the HDV RNA was processed as if it were a host mRNA polymerase II transcript, although this did not necessarily indicate that HDV RNA was transcribed with this enzyme. (+info)
Positive selection of hepatitis delta antigen in chronic hepatitis D patients.
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Liver disease may become ameliorated in some patients with chronic hepatitis D virus (HDV) infection. We present here a study based on longitudinal sampling to investigate the viral dynamics in chronic HDV infection. We examined the HDV variants from different time points, especially those before and after the elevation of serum aminotransferase levels. The datasets from each patient were tested for positive selection by using maximum-likelihood methods with heterogeneous selective pressures along the nucleotide sequence. An average of 4.9%, ranging from 3.1 to 6.8%, of the entire delta antigen sites was regulated by a diversifying selection. Most of the positively selected sites were associated with immunogenic domains. Likelihood ratio tests revealed a significant fitness of positive selection over neutrality of the hepatitis delta antigen gene in all patients. We further adapted a neural network method to predict potential cytotoxic T ligand epitopes. Among the HLA-A*0201 cytotoxic T ligand epitopes, three consistent epitopes across all three genotypes were identified: amino acids (aa) 43 to 51, 50 to 58, and 114 to 122. Three patients (60%) had sites evolving under positive selection in the epitope from aa 43 to 51, and four patients (80%) had sites evolving under positive selection in the epitope from aa 114 to 122. The discovery of immunogenic epitopes, especially cytotoxic-T-lymphocyte ligands, associated with chronic HDV infection may be crucial for further development of novel treatments or designs in vaccine for HDV superinfection. (+info)
Immunoblot analysis demonstrates that the large and small forms of hepatitis delta virus antigen have different C-terminal amino acid sequences.
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Antisera to a peptide representing the extreme carboxy terminus of the hepatitis delta virus antigen (HDAg) open reading frame (residues 197 to 211) recognized only the large (p27 delta) and not the small (p24 delta) form of HDAg in immunoblots of infected liver extracts, thereby providing direct proof that p27 delta and p24 delta differ in their carboxyl-terminal sequence and that p27 delta results from mutation within the stop codon terminating translation of p24 delta. Reactions with other peptide antisera demonstrated that multiple smaller virus-specified proteins were carboxy-terminally truncated forms of HDAg, and immunoprecipitation studies suggested that different forms of HDAg were present as heterologous complexes within the liver extract. (+info)
Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.
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Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex. (+info)
Large hepatitis delta antigen is a novel clathrin adaptor-like protein.
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Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin. (+info)
Evaluation of commercial enzyme immunoassays for detection of hepatitis delta antigen and anti-hepatitis delta virus (HDV) and immunoglobulin M anti-HDV antibodies.
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Panels of hepatitis B virus surface antigen-positive sera from drug abusers were used to evaluate 14 commercial enzyme immunoassays from six companies for detecting hepatitis delta virus (HDV) markers. For detecting hepatitis delta virus antigen (HDAg), the Wellcome, Pasteur and Noctech assays had 100% sensitivity for all 42 HDAg-positive serum specimens that were confirmed in-house; the Organon reagents gave 59.5% sensitivity without detergent and 64.3% sensitivity with detergent, but there were 14 discrepant results with and without detergent. The Sorin assay detected HDAg in only 10 of the positive samples (23.8% sensitivity). For the detection of antibody to HDV (anti-HDV) all six commercial enzyme immunoassays were reactive with all 36 anti-HDV-positive specimens that were confirmed in-house. There were no false-positive results with the Wellcome, Noctech, or Sorin assay, but one specimen was false positive by the Organon assay. One HDAg-positive specimen gave a false anti-HDV-positive result in the Abbott assay and an equivocal result in the Pasteur assay (97.8% specificity). For the detection of immunoglobulin M anti-HDV, the Wellcome, Noctech, and Sorin assays agreed for the 38 positives confirmed in-house, except for one false negative with the Sorin test. We conclude that there has been a substantial improvement over previously evaluated assays in sensitivity and specificity of commercial assays for anti-HDV detection, and the sensitivities of immunoglobulin M anti-HDV assays are also comparable. However, there are still major differences in sensitivity among some assays for HDAg detection. (+info)