Rapid Reports Multiple Targets for Suppression of RNA Interference by Tomato Aspermy Virus Protein 2B † Umar Jan Rashid, ‡ Jan Hoffmann, § Bernhard Brutschy, § Jacob Piehler, | and Julian C.-H. Chen* ,‡ Institute of Biophysical Chemistry, Institute of Physical and Theoretical Chemistry, and Institute of Biochemistry, Goethe UniVersity Frankfurt, Max-Von-Laue-Strasse 9, 60438 Frankfurt, Germany ReceiVed July 9, 2008; ReVised Manuscript ReceiVed September 30, 2008 ABSTRACT: Viral suppressors of RNA interference (RNAi) appear to have evolved as a response to this innate genomic defense. We report the nucleic acid binding properties of the CucumoVirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B). Using total internal reflection fluorescence spectroscopy (TIRFS), we show that TAV 2B binds double-stranded RNA corresponding to siRNAs and miRNAs, as well as single-stranded RNA oligonucleotides. A number of positively charged residues between amino acids 20 and 30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B, causing it to form a primarily helical structure and a 4:2 protein-RNA complex. RNA interference (RNAi), an ancient mechanism for gene silencing triggered by recognition of dsRNA, is thought to have emerged as a way of safeguarding the genome against mobile genetic elements and the infection of viruses, and thus is a way of maintaining genomic integrity (1-5). Therefore, it is not surprising that viruses have evolved different strategies for suppressing the host RNAi response in the form of viral suppressor protein. These viral suppres- sors are widespread, having been identified in a number of different viral families. Not surprisingly, they generally share little sequence homology with one another, although they appear to exist as oligomers built upon an ∼100-200-amino acid protomer. Tomato aspermy virus, a member of the Cucumoviruses, encodes protein 2B (TAV 2B, 95 amino acids, ∼11.3 kDa) that acts as an RNAi suppressor. Intriguingly, a similar genomic arrangement is seen in RNAi suppressors in the Nodaviruses, a family of viruses that can infect both plants and animals, such as Flock house virus b2 (FHV b2). The 2B and b2 proteins are both derived from a frame-shifted open reading frame (ORF) within the RNA polymerase gene (6). In spite of this genomic similarity, the 2B and b2 proteins share little sequence identity, and it is not well understood how the CucumoVirus 2B proteins suppress RNAi. To address this question, we report the characterization and oligonucleotide binding properties of TAV 2B and discuss possible modes of suppression of RNAi by this protein. Full-length TAV 2B expressed poorly and was marginally soluble. On the basis of a sequence alignment with other 2B suppressors of the Cucumoviruses, and previous domain swapping experiments, a truncated construct consisting of amino acids 1-71 (TAV71) was expressed and purified (7, 8). Affinity-purified TAV71 migrates as a monomeric species on a Superdex 200 gel filtration column, at a physiological salt (150 mM) concentration (data not shown). The high percentage of basic residues in the primary sequence suggested that it may bind nucleic acids. To investigate the potential oligonucleotide binding properties of TAV71, total internal reflection fluorescence spectroscopy (TIRFS) was used to probe the binding of fluorescently labeled oligonucleotide substrates to TAV71 in real time. For this purpose, TAV71 is immobilized onto a sensor chip through its His tag (9, 10). The fluorescently labeled oligonucleotide is excited by the evanescent wave emanating from the surface. Thus, a signal is recorded only when the labeled molecule is bound to protein immobilized on the surface (Figure 1 of the Supporting Information). Suppression of RNAi may occur through a direct interac- tion with siRNAs. Plants have a distribution of different siRNA lengths; shorter species (21-23 nucleotides) are involved in the RNAi response, while longer siRNAs (25-27 nucleotides) are associated with transcriptional silencing and the spread of silencing. To determine if TAV71 binds siRNAs, and if so, whether there was a length preference for this recognition, fluorescently labeled siRNAs of 21, 25, and 27 nucleotides were probed for binding to TAV71 immobilized on a Ni-NTA chip. At a given protein concen- tration on the surface, the observed fluorescence amplitude is ∼3-fold higher for the 21-nucleotide siRNA (400 mV) than for 25- and 27-nucleotide siRNAs (110 and 130 mV, respectively). As a control, no appreciable fluorescence signal is observed in the absence of immobilized protein (Figure 2 of the Supporting Information). Thus, TAV71 recognizes siRNAs and preferentially binds 21-nucleotide siRNA com- pared to 25- and 27-nucleotide siRNAs (Figure 1a). The † This work was funded by DFG SFB 579 and the Hessian Ministry for Science and Culture. * To whom correspondence should be addressed. Phone: +49 (0) 69 798 29641. Fax: +49 (0) 69 798 29632. E-mail: chen@ chemie.uni-frankfurt.de. ‡ Institute of Biophysical Chemistry. § Institute of Physical and Theoretical Chemistry. | Institute of Biochemistry. Biochemistry 2008, 47, 12655–12657 12655 10.1021/bi801281h CCC: $40.75  2008 American Chemical Society Published on Web 11/06/2008