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Journal of Virology, July 2008, p. 6911-6926, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00702-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein{triangledown}

Zhenghe Li, Daniel Barajas, Tadas Panavas, David A. Herbst, and Peter D. Nagy*

Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546

Received 28 March 2008/ Accepted 1 May 2008

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, Lexington, KY 40546. Phone: (859) 257-7445, ext. 80726. Fax: (859) 323-1961. E-mail: pdnagy2{at}uky.edu

{triangledown} Published ahead of print on 7 May 2008.

Journal of Virology, July 2008, p. 6911-6926, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00702-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Barajas, D., Li, Z., Nagy, P. D. (2009). The Nedd4-Type Rsp5p Ubiquitin Ligase Inhibits Tombusvirus Replication by Regulating Degradation of the p92 Replication Protein and Decreasing the Activity of the Tombusvirus Replicase. J. Virol. 83: 11751-11764 [Abstract] [Full Text]  
  • Wang, R. Y.-L., Stork, J., Nagy, P. D. (2009). A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes. J. Virol. 83: 3276-3287 [Abstract] [Full Text]  
  • Pogany, J., Stork, J., Li, Z., Nagy, P. D. (2008). In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70. Proc. Natl. Acad. Sci. USA 105: 19956-19961 [Abstract] [Full Text]  


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