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Journal of Virology, March 2006, p. 3000-3008, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.3000-3008.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biological Relevance of a Stable Biochemical Interaction between the Tombusvirus-Encoded P19 and Short Interfering RNAs

Rustem Omarov,1 Kim Sparks,1 Lindsay Smith,1 Jelena Zindovic,1,{dagger} and Herman B. Scholthof1,2*

Department of Plant Pathology and Microbiology,1 Intercollegiate Faculty of Virology, Texas A&M University, 2132 TAMU, College Station, Texas 778432

Received 6 September 2005/ Accepted 17 December 2005

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex ~21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for ~21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.

* Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Texas A&M University, 2132 TAMU, College Station, TX 77843. Phone: (979) 862-1495. Fax: (979) 845-6483. E-mail: herscho{at}tamu.edu.

{dagger} Present address: Department of Plant Protection, Biotechnical Institute, University of Montenegro, 81000 Podgorica, Serbia and Montenegro.

Journal of Virology, March 2006, p. 3000-3008, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.3000-3008.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hsieh, Y.-C., Omarov, R. T., Scholthof, H. B. (2009). Diverse and Newly Recognized Effects Associated with Short Interfering RNA Binding Site Modifications on the Tomato Bushy Stunt Virus P19 Silencing Suppressor. J. Virol. 83: 2188-2200 [Abstract] [Full Text]  
  • Valli, A., Dujovny, G., Garcia, J. A. (2008). Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus. J. Virol. 82: 974-986 [Abstract] [Full Text]  
  • Scholthof, H. B. (2007). Heterologous Expression of Viral RNA Interference Suppressors: RISC Management. Plant Physiol. 145: 1110-1117 [Full Text]  
  • Omarov, R. T., Ciomperlik, J. J., Scholthof, H. B. (2007). RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex. Proc. Natl. Acad. Sci. USA 104: 1714-1719 [Abstract] [Full Text]  


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