Chaperone-Mediated in vitro Assembly of Polyomavirus Capsids

The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell n...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 100; no. 18; pp. 10477 - 10482
Main Authors:	Chromy, Laura R., Pipas, James M., Garcea, Robert L.
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 02-09-2003 National Acad Sciences
Subjects:	Adenosine Triphosphate - metabolism Biological Sciences Calcium Capsid Capsid proteins Capsid Proteins - physiology Deoxyribonucleic acid DNA Escherichia coli Proteins Gel chromatography HSP70 Heat-Shock Proteins - physiology Microbiology Polyomavirus Polyomavirus - physiology Proteins Ungulates Viral proteins Virions Virus Assembly Viruses
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Summary:	The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for ≈70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperonemediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 To whom correspondence should be addressed at: University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box C229, Denver, CO 80262. E-mail: bob.garcea@uchsc.edu. Abbreviations: VP1, viral protein 1; VP1 + VP3, VP1 pentamer with VP3; VLP, virus-like particle; LgT, large T-antigen; TEM, transmission electron microscopy; SV40, simian virus 40; hsc70, ≈70-kDa heat shock cognate protein; hsp70, ≈70-kDa heat shock protein. This paper was submitted directly (Track II) to the PNAS office. Edited by Timothy A. Springer, Harvard Medical School, Boston, MA, and approved July 8, 2003
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.1832245100