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Journal of Virology, January 2008, p. 974-986, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01664-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus ^,

Adrian Valli, Gabriela Dujovny, and Juan Antonio García^*

Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain

Received 31 July 2007/ Accepted 25 October 2007

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.

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* Corresponding author. Mailing address: Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain. Phone: 34-915854535. Fax: 34-915854506. E-mail: jagarcia{at}cnb.uam.es

Published ahead of print on 7 November 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, January 2008, p. 974-986, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01664-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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