In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70

To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-12, Vol.105 (50), p.19956-19961
Main Authors: Pogany, Judit, Stork, Jozsef, Li, Zhenghe, Nagy, Peter D
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Biological Sciences
cell free system
Cells
Escherichia coli
Genetic recombination
Guanosine Triphosphate - metabolism
Heat shock proteins
Host-Pathogen Interactions
host-pathogen relationships
HSP70 Heat-Shock Proteins - genetics
HSP70 Heat-Shock Proteins - metabolism
in vitro studies
Integration host factors
Mutation
protein-protein interactions
Ribonucleic acid
Ribonucleotides
RNA
RNA Replicase - metabolism
RNA, Viral - metabolism
RNA-directed RNA polymerase
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Tomato bushy stunt virus
Tomatoes
Tombusvirus
Tombusvirus - enzymology
Tombusvirus - genetics
Tombusvirus - physiology
Viral Proteins - metabolism
Viral RNA
Virology
Virus Replication
Viruses
Yeast
Yeast extract
Yeasts
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Summary:To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which were obtained from E. coli, the viral RNA replicon, rATP, rGTP, and a yeast cell-free extract. The in vitro assembly of the replicase took place in the membraneous fraction of the yeast extract, in which the viral replicase-RNA complex became RNase- and proteinase-resistant. The assembly of the replicase complex required the heat shock protein 70 (Hsp70 = yeast Ssa1/2p) present in the soluble fraction of the yeast cell-free extract. The assembled TBSV replicase performed a complete replication cycle, synthesizing RNA complementary to the provided RNA replicon and using the complementary RNA as template to synthesize new TBSV replicon RNA.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0810851105