JVI Accepts, published online ahead of print on 7 November 2007

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J. Virol. doi:10.1128/JVI.01664-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Protease activity, self interaction and siRNA 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

^* To whom correspondence should be addressed. Email: jagarcia{at}cnb.uam.es.

The RNA silencing pathway mediated by 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 has been reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, HCPro, has a duplicated P1 coding sequence, and 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 its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif placed at the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration FPLC and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and 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 in the RNA-induced silencing complex.

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