RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs (dsRNAs) and provide the sequence specificity for degradation of cognate RNA molecules. In plants, RNA silencing plays a key role in developmental processes and in control of virus replication. It has been shown that many plant viruses encode proteins, denoted RNA silencing suppressors, that interfere with this antiviral response. Although RNA silencing has been shown to occur in vertebrates, no relationship with inhibition of virus replication has been demonstrated to date. Here we show that the NS1 protein of human influenza A virus has an RNA silencing suppression activity in plants, similar to established RNA silencing suppressor proteins of plant viruses. In addition, NS1 was shown to be capable of binding siRNAs. The data presented here fit with a potential role for NS1 in counteracting innate antiviral responses in vertebrates by sequestering siRNAs. INTRODUCTIONSeveral observations made in plants such as transgeneinduced co-suppression of genes (Van der Krol et al., 1990;Napoli, 1990), post-transcriptional gene silencing (English et al., 1996), RNA-mediated virus resistance (Lindbo & Dougherty, 1992;de Haan et al., 1992), virus-induced gene silencing, and more recently in other organisms such as quelling in fungi (Cogoni & Macino, 1997) and RNA interference (RNAi) in nematodes, insects and mammals (Elbashir et al., 2001; Fire et al., 1998;Tuschl et al., 1999), have turned out to rely on a similar molecular process. This process, now referred to as RNA silencing, is induced by overexpressed double-stranded RNA (dsRNA) molecules and involves sequence-specific RNA degradation in the cytoplasm of eukaryotic cells (Sharp, 2001). The degradation products of this process, which is catalysed by an enzyme first identified in flies as DICER (Bernstein et al., 2001), are RNAs of 21-25 nt.Two functional classes of these molecules produced by DICER cleavage have thus far been identified: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The presence of these molecules is regarded as a hallmark of RNA silencing (Hamilton & Baulcombe, 1999). In plants, miRNAs seem to be predominantly involved in targeted mRNA degradation of transcription factors that play a role in development (Llave et al., 2002; Palatnik et al., 2003), while siRNAs recruit specific proteins to form the RNAinduced silencing complex (RISC) and initiate sequencespecific degradation of target RNAs, such as viral RNAs (reviewed by Vaucheret & Fagard, 2001;Zamore, 2002).The siRNA-mediated RNA silencing machinery has been suggested to play different roles in different organisms. In plants, its major function seems to be providing antiviral defence at the nucleic acid level. Indeed, Arabidopsis mutants exhibiting impaired RNA silencing show enhan...

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNAinduced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.

Recent genome-wide surveys showed that acetylation of H3K9 and H3K27 is correlated with gene activation during deetiolation of Arabidopsis thaliana seedlings, but less is known regarding changes in the histone status of repressed genes. Phytochrome A (phyA) is the major photoreceptor of deetiolation, and phyA expression is reversibly repressed by light. We found that in adult Arabidopsis plants, phyA activation in darkness was accompanied by a significant enrichment in the phyA transcription and translation start sites of not only H3K9/14ac and H3K27ac but also H3K4me3, and there was also moderate enrichment of H4K5ac, H4K8ac, H4K12ac, and H4K16ac. Conversely, when phyA expression was repressed by light, H3K27me3 was enriched with a corresponding decline in H3K27ac; moreover, demethylation of H3K4me3 and deacetylation of H3K9/14 were also seen. These histone modifications, which were focused around the phyA transcription/ translation start sites, were detected within 1 h of deetiolation. Mutant analysis showed that HDA19/HD1 mediated deacetylation of H3K9/14 and uncovered possible histone crosstalk between H3K9/14ac and H3K4me3. Neither small RNA pathways nor the circadian clock affected H3 modification status of the phyA locus, and DNA methylation was unchanged by light. The presence of activating and repressive histone marks suggests a mechanism for the rapid and reversible regulation of phyA by dark and light.

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