An intramolecular disulfide bridge between Cys-7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus.
Hepatitis B virus, the prototypic member of the Hepadnaviridae, is a small enveloped DNA virus that replicates via reverse transcription. Efficient usage of its compact 3.2-kb genome is exemplified by the pre-C/C gene from which two proteins with largely overlapping primary sequences but distinctly different properties are synthesized: the self-assembling core protein p21c (hepatitis B core antigen [HbcAg]) and the secretory, nonparticulate protein p17e (hepatitis B e antigen [HbeAg]). Mature p17e carries a 10-amino-acid N-terminal extension with a Cys residue (Cys-7). Using transient transfection of a human liver cell line with constructs expressing wild-type p17 or a series of Cys mutants of p17, we show that Cys-7 forms an intramolecular S-S bond to Cys61, which in assembly-competent core proteins is available for intermolecular disulfide bonds between two neighboring subunits. Removal of the Cys-7/Cys61 bond by mutating either residue has differential effects: in the absence of Cys-7, secretion is relatively efficient and independent of Cys61; however, the molecules are exported as homodimers exhibiting both HBe and HBc antigenicity. In the absence of Cys61, the nonpaired Cys-7 interferes with secretion efficiency. The amino acid sequence flanking Cys-7 also contributes to the formation of the proper intramolecular S-S bond. These results suggest that the Cys-7/Cys61 bond imposes on p17e a conformation that is critical for its secretion and distinct biophysical and antigenic properties. This mechanism adds selective disulfide formation to the repertoire of hepatitis B virus for efficient use of its tiny genome.
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
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Nassal
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Rieger
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004220:Disulfides; D006513:Hepatitis B e Antigens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection
D004220:Disulfides; D006513:Hepatitis B e Antigens; D003545:Cysteine