Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.
The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.
D T
DT
Wong
N
N
Nath
J J
JJ
Sninsky
D004274:DNA, Recombinant; D000939:Epitopes; D005814:Genes, Viral; D008040:Genetic Linkage; D006023:Glycoproteins / Q000235:genetics; D006510:Hepatitis B Antibodies / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D008970:Molecular Weight; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology
D004274:DNA, Recombinant; D000939:Epitopes; D006023:Glycoproteins; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D014764:Viral Proteins