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Chaperone-mediated in vitro assembly of Polyomavirus capsids
Laura R Chromy1, James M Pipas, Robert L Garcea
1Section of Pediatric Oncology and Molecular Biology Program, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Summary
Cellular chaperones, like heat shock protein 70 (hsp70), regulate polyomavirus capsid assembly. These hsp70 systems can assemble uniform polyomavirus VP1 capsids in vitro, mimicking in vivo processes.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Polyomavirus coat protein (VP1) self-assembles into polymorphic structures in vitro.
- In vivo, polyomavirus assembly is nuclear and produces uniform virions.
- Cellular heat shock cognate protein 70 (hsc70) interacts with VP1 during infection.
Purpose of the Study:
- To investigate the role of heat shock protein 70 (hsp70) family chaperones in polyomavirus capsid assembly.
- To determine if hsp70 systems can recapitulate in vivo-like capsid formation in vitro.
Main Methods:
- Recombinant VP1 expressed in E. coli was purified.
- Interactions between VP1 and prokaryotic DnaK were analyzed.
- In vitro assembly assays were performed using prokaryotic (DnaK, DnaJ, GrpE) and eukaryotic (hsc70) chaperone systems with and without ATP and calcium.
Main Results:
- Prokaryotic DnaK copurified with the VP1 C-terminal domain and inhibited calcium-induced assembly.
- The prokaryotic hsp70 system (DnaK, DnaJ, GrpE) with ATP assembled uniform VP1 capsids without calcium.
- The eukaryotic hsc70 system also catalyzed uniform capsid assembly.
Conclusions:
- Polyomavirus capsid assembly can be accurately reproduced in vitro using hsp70 chaperone systems.
- Cellular hsp70 chaperones play a crucial role in regulating the fidelity and location of in vivo virion assembly.