A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.
We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.
Lingappa
J R
JR
Department of Physiology, University of California at San Francisco 94143.
Martin
R L
RL
Wong
M L
ML
Ganem
D
D
Welch
W J
WJ
Lingappa
V R
VR
D000595:Amino Acid Sequence; D002213:Capsid / Q000378:metabolism*; D002474:Cell-Free System; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D003600:Cytosol / Q000378:metabolism; D019286:Endopeptidase K; D006367:HeLa Cells; D006515:Hepatitis B virus / Q000254:growth & development* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D014758:Viral Core Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D046911:Macromolecular Substances; D011506:Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins