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Journal of Virology, November 2009, p. 11751-11764, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.00789-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Daniel Barajas, Zhenghe Li, and Peter D. Nagy*

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

Received 19 April 2009/ Accepted 3 September 2009

Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82:6911-6926, 2008). To further our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with p33 and the central portion of p92pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92pol, while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92pol. We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92pol reduces the stability of p92pol, with consequent inhibition of TBSV replicase activity.

* 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 16 September 2009.

Journal of Virology, November 2009, p. 11751-11764, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.00789-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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