RNA silencing or post-transcriptional gene silencing (PTGS) in plants is known as a defense system against virus infection. Several plant viruses have been shown to encode an RNA silencing suppressor. Using a green £uorescent proteinbased transient suppression assay, we show that NSs protein of Tomato spotted wilt virus (TSWV) has RNA silencing suppressor activity. TSWV NSs protein suppressed sense transgene-induced PTGS but did not suppress inverted repeat transgene-induced PTGS. TSWV NSs protein is the ¢rst RNA silencing suppressor identi¢ed in negative-strand RNA viruses.

P ositive-strand RNA viruses cause numerous human, animal, and plant diseases including encephalitis, hemorrhagic fevers, and hepatitis. Hepatitis C virus, e.g., chronically infects hundreds of millions of people, leading to progressive liver damage and cancer. Such positive-strand RNA viruses encapsidate messenger-sense RNA genomes, replicate through negative-strand RNA intermediates with no natural DNA phase, and share fundamental similarities in RNA replication. At the beginning of infection, the viral genomic RNA is first translated, yielding RNA replication factors that must recruit the genomic RNA out of translation for 3Ј to 5Ј copying by viral polymerase (1-3). The resulting RNA replication complexes are associated with intracellular membranes, although the nature and functions of this association are poorly understood (4-6).Genetic and biochemical results suggest that RNA virus replication involves unidentified host factors (7-10). Identifying and characterizing the relevant host factors is thus an important frontier for understanding and controlling viral replication as well as host range and pathology and should also illuminate important host cell pathways. To facilitate such studies, our laboratory identified two higher eukaryotic viruses able to replicate in the genetically tractable yeast Saccharomyces cerevisiae (11,12). One of these, brome mosaic virus (BMV), is a member of the alphavirus-like superfamily of human-, animal-, and plant-infecting positive-strand RNA viruses (13).The BMV genome is divided among three 5Ј capped RNAs (Fig. 1A). RNA1 and RNA2 encode replication factors 1a and 2a, which share conserved domains with all members of alphavirus-like superfamily. 1a contains domains implicated in RNA helicase and RNA capping functions (14, 15), and 2a contains an RNA-dependent RNA polymerase domain (13). 1a and 2a colocalize in an endoplasmic reticulum-associated replication complex that is the site of BMV-specific RNA synthesis (4, 5). RNA3 encodes cell-to-cell movement and coat proteins that direct systemic infection in the natural plant hosts of BMV but are dispensable for RNA replication. Coat protein is not translated from RNA3 but only from a subgenomic mRNA, RNA4 (11, 16). Expression of the coat gene or genes replacing it thus requires RNA3 replication to produce negative-strand RNA3, followed by subgenomic mRNA synthesis (Fig. 1 A). Like RNAs of hepatitis C virus and many other RNA viruses, BMV RNAs lack the 3Ј poly(A) of cellular mRNAs. Instead, the BMV RNAs have a tRNA-like structure that interacts with multiple tRNA-specific cellular enzymes (17,18).In yeast expressing 1a and 2a, RNA3 or RNA3 derivatives introduced by transfection or in vivo transcription are replicated and synthesize subgenomic RNA4 (Fig. 1 A). This yeast system reproduces all known features of BMV RNA replication in natural plant hosts, including localization to the endoplasmic reticulum; dependence on 1a, 2a, and the same cis-acting RNA signals; similar ratios of positive to negative-strand RNA; and other featu...

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3 untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3 UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 533 decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3-side RNA sequences against 533 decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae. Many lines of recent evidence indicate that noncodingRNAs including microRNAs and small interfering RNAs play an important role in the control of gene expression in diverse cellular processes and in defense responses against molecular parasites such as viruses and transposons. Viruses also use many different types of RNAs in trans for regulating the expression of their own genomes or host genomes temporally and spatially to ensure efficient virus proliferation and/or latency in host cells. These RNAs include subgenomic RNAs (sgRNAs), viral genomic RNA itself, and many types of noncoding viral RNAs.For example, the adenovirus virus-associated RNAs (VA RNAs) (23) are small noncoding RNA transcripts. They inhibit the activation of RNA-induced protein kinase and thereby interfere with the activation of the interferon-induced cellular antiviral defense systems (38). VA RNAs also interfere with RNA interference pathways by acting as substrates for Dicer and suppressing the activity of Dicer probably involved in cellular antiviral mechanisms (2, 55). Epstein-Barr virus-encoded RNAs (EBERs) (56) inhibit RNA-induced protein kinase as VA RNAs (38). They also are known to encode microRNAs, which are thought to work for persistent infection (28). On the other hand, recently, EBERs have been reported to be recognized by RIG-I, a cytosolic protein with a DexD/H box RNA helicase domain that recognizes viral RNA in mammalian cells, and to activate signaling to induce type I interferon (35). Thus, associations of viral small RNAs with virus infection are complicated.sgRNAs also fun...

RNA interference (RNAi) is a post-transcriptional generegulatory mechanism that operates in many eukaryotes. RNAi is induced by double-stranded RNA (dsRNA) and is mainly involved in defence against transposons and viruses. To counteract RNAi, viruses have RNAi suppressors. Here we show a novel mechanism of RNAi suppression by a plant virus Red clover necrotic mosaic virus (RCNMV). To suppress RNAi, RCNMV needs multiple viral components, which include viral RNAs and putative RNA replicase proteins. A close relationship between the RNA elements required for negative-strand RNA synthesis and RNAi suppression suggests a strong link between the viral RNA replication machinery and the RNAi machinery. In a transient assay, RCNMV interferes with the accumulation of small-interfering RNA (siRNAs) in RNAi induced by a hairpin dsRNA and it also interferes with microRNA (miRNA) biogenesis. An Arabidopsis dcl1 mutant showed reduced susceptibility to RCNMV infection. Based on these results, we propose a model in which, to replicate, RCNMV deprives the RNAi machinery of Dicer-like enzymes that are involved in both siRNA and miRNA biogenesis.