title: Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo.
abstract: Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill 27599-7030.
authors.forename: J G
authors.initials: JG
authors.lastname: Wang
authors.forename: S M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism; D000939:Epitopes; D005976:Glutaral; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D003698:Hepatitis Delta Virus / Q000737:chemistry* / Q000187:drug effects; D035923:Hepatitis delta Antigens; D006801:Humans; D008099:Liver / Q000737:chemistry; D046911:Macromolecular Substances; D008392:Marmota / Q000382:microbiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D014771:Virion / Q000737:chemistry
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D003432:Cross-Linking Reagents; D004220:Disulfides; D000939:Epitopes; D006146:Guanidines; D035923:Hepatitis delta Antigens; D046911:Macromolecular Substances; D012367:RNA, Viral; C442244:hepatitis delta virus large antigen; D019791:Guanidine; D005976:Glutaral