The surface antigen of hepatitis B virus comprises a nested set of small (S), middle (M), and large (L) proteins, all of which are partially glycosylated in their S domains. The pre-S2 domain, present only in M and L proteins, is further N-glycosylated at Asn-4 exclusively in the M protein. Since the pre-S2 N-glycan appears to play a crucial role in the secretion of viral particles, the M protein may be considered as a potential target for antiviral therapy. For characterization of the pre-S2 glycosylation, pre-S2 (glyco)peptides were released from native, patient-derived hepatitis B virus subviral particles by tryptic digestion, separated from remaining particles, purified by reversed-phase high performance liquid chromatography, and identified by amino acid and N-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion exchange chromatography, methylation analysis, and on target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides. In addition, the pre-S2 domain of M protein, but not that of L protein, was found to be partially O-glycosylated by a Gal(␤1-3)GalNAc␣-, Neu5Ac(␣2-3)Gal(␤1-3)GalNAc␣-, or GalNAc␣-residue. The respective O-glycosylation site was assigned to Thr-37 by digestion with carboxypeptidases in combination with MALDI-TOF-MS and by quadrupole time-of-flight electrospray mass spectrometry. Analytical data further revealed that about 90% of M protein is N-terminally acetylated. Hepatitis B virus (HBV),1 belonging to the virus family hepadnaviridae, is an important etiological agent of acute and chronic liver disease (1, 2). Chronic HBV infection may lead to liver cirrhosis and hepatocellular carcinoma, which result in about 1 million deaths per year worldwide. The virus replicates in the liver and is secreted in large amounts of up to 10 10 particles/ml into the blood (3). In addition to 42-nm DNA containing virions, infected hepatocytes produce subviral, noninfectious 22-nm spherical or filamentous particles in vast excess. The envelopes of virions and subviral particles contain varying amounts of three related HBV-encoded (glyco)protein species termed large (L), middle (M), and small (S) proteins, which are together referred to as HBV surface antigen (HBsAg). S protein is the major component of virions and both spherical and filamentous HBsAg particles, while filaments and virions contain more M and, in particular, more L proteins than spheres (4, 5). All envelope proteins are produced from a single open reading frame (see Fig. 1A) by the use of three different translation start sites, dividing this open reading frame into three domains: the amino-terminal pre-S1 domain, which occurs exclusively in the L protein; the pre-S2 domain, which is present in both M and L proteins and forms the amino-terminal end of the M protein; and the S domain, which is common to S, M, an...

Since the discovery of woodchuck hepatitis virus (WHV) in 1978, the virus and its host, the American woodchuck, have been studied and used as the most suitable model for human hepatitis B virus infection. WHV is closely related to the human virus, having strong similarities in morphology, genome structure and gene products, replication, epidemiology, the course of infection and in the development of illness and hepatocellular carcinoma. Because of this high homology, the woodchuck model is used for many studies for the development of new vaccines, therapeutic vaccination and antiviral agents. In addition, the woodchuck system is used for investigation of molecular mechanisms of the viral life cycle, the mechanisms of carcinogenesis and cell infection.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV)infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide 1-20 , peptide 100-119 , and peptide 112-131 . Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide 97-110 . Establishment of polyclonal T-cell lines with WHcAg or peptide 97-110 revealed reciprocal stimulation by peptide 97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide [97][98][99][100][101][102][103][104][105][106][107][108][109][110] or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide 97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection. on July 16, 2020 by guest http://jvi.asm.org/ Downloaded from MATERIALS AND METHODS Woodchucks. Adult WHV-negative woodchucks trapped in the state of New York and chronically WHV-infected woodchucks trapped in the state of Delaware were purchased from North Eastern Wildlife (Ithaca, N.Y.).Woodchucks were defined WHV negative if no WHV DNA and no antibodies against surface protein (anti-WHs) and core protein (anti-WHc) were detectable in the sera. Sera from chronically WHV-infected woodchucks were positive for WHV DNA, surface protein (WHsAg), and anti-WHc but negative for anti-WHs. Experimental infection of WHV-negative woodchucks was performed by intravenous (i.v.) inoculation with 10 5 woodchuck 50% infective doses (ID 50 ) of a titrated WHV pool (39). The sera of acutely WHV-infected woodchucks, obtained 4 to 6 weeks after inoculation, were positive for WHV DNA and WHsAg. Sera obtained 10 to 20 weeks after inoculation (postacute phase of WHV infection) were negative for WHV DNA and WHsAg and positive for anti-WHs and anti-WHc.Immunization with rWHcAg or WHcAg-derived peptides. Four WHV-negative woodchucks w...

The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion-exchange chromatography, methylation analysis and on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides in all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(a2-3)Gal(b1-3)GalNAca-or Gal(b1-3)GalNAca-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.

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