Mutational analysis of hepatitis B surface antigen particle assembly and secretion.
Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.
Bruss
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Department of Microbiology and Immunology, University of California Medical Center, San Francisco 94143-0502.
Ganem
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D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005814:Genes, Viral; D005914:Globins / Q000737:chemistry; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D021382:Protein Sorting Signals / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D015678:Viral Structural Proteins / Q000235:genetics
D006514:Hepatitis B Surface Antigens; D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D005914:Globins