RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the Bunyaviridae. Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS S gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS S -expressing leaf sectors suggests that the tospoviral NS S protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.RNA silencing involves a sequence-specific degradation which is induced by overabundant RNA and by doublestranded RNA (dsRNA) molecules and which can target transgenes as well as homologous endogenous genes. RNA silencing was first described for plants (35,50) and over recent years has been described for other organisms, where it is also referred to as cosuppression, posttranscriptional gene silencing (17), or RNA-mediated virus resistance (3,11,30) in plants, quelling in fungi (9), or RNAi in animals (19). Building blocks of the gene-silencing pathway proved to have remarkable similarities in the different organisms and hence suggest an ancient role of gene silencing in pathogen resistance or development (10,25,53). One of the key intermediary elements in the RNA silencing pathway is dsRNA, which is recognized by a dsRNAspecific nuclease (5) to yield small (21 to 23 nucleotides) short interfering RNAs (siRNAs) (21). These siRNAs subsequently serve as guides for cleavage of homologous RNA molecules. In plants, versions of transgenes that produce dsRNA molecules have been shown to be very potent activators of RNA silencing (47). As all RNA viruses replicate through formation of dsRNA intermediates, these are potential targets of the RNA silencing mechanism. Indeed, antiviral RNA silencing has been shown to occur in nature and has been proposed as a natural defense mechanism protecting plants against viruses, resulting in resistance (1, 43).To counteract the RNA silencing mechanism of their host, plant viruses have developed ways to evade or neutralize this response. Over recent years, RNA silencing-inhibiting proteins have been identified in several plant viruses. Among the best-studied examples are the helper component-proteinase (HC-Pro) of the potyvirus Potato virus Y (PVY) and the 2b protein of Cucumber mosaic virus (CMV) (7, 40). Other plus-strand RNA (and some DNA) viruses also have been found to suppress gene silencing, and ...

The complete nucleotide sequence of the S RNA of tomato spotted wilt virus (TSWV) was determined. The RNA is 2916 nucleotides long and has an ambisense coding strategy. The sequence contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted Mr of 52"4K and one in the viral complementary sense which encodes the viral nucleocapsid protein of Mr 28"8K. Both proteins are expressed by translation of two subgenomic RNA species that possibly terminate at a long stable hairpin structure, located at the intergenic region. The structure of this RNA segment resembles that of the arthropod-borne phleboviruses (family Bunyaviridae). The absence of significant sequence homology between TSWV and bunyaviruses infecting animals suggests that TSWV should be considered as a representative of a new genus within the Bunyaviridae.

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...