The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.
Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.
Winokur
P L
PL
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City.
McLinden
J H
JH
Stapleton
J T
JT
D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006508:Hepatitis Antibodies / Q000276:immunology; D006507:Hepatovirus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009841:Oligonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014461:Ultracentrifugation; D014616:Vaccinia virus
D006508:Hepatitis Antibodies; D009841:Oligonucleotides; D011506:Proteins; D011994:Recombinant Proteins