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.

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...

In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response.

Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts.Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.Plant viruses encode proteins that control their movement from cell to cell. These proteins are called movement proteins (MPs) and interact with the normal symplastic connections between plant cells, the plasmodesmata, by modifying the plasmodesmal structure and function. Consequently, the highly regulated passage of small molecules through plasmodesmata is altered to allow the passage of large nucleoprotein complexes containing the viral genome (8, 28).Some viruses, including Tobacco mosaic virus (TMV) and Red clover necrotic mosaic virus, do not require the viral coat protein (CP) for cell-to-cell movement. The MPs of these viruses have a nucleic acid binding activity (10, 37), and an MP-RNA nucleoprotein complex is thought to pass through the modified plasmodesmata to adjacent uninfected cells (8,26). Other viruses do not move from cell to cell in the absence of viral CP. Cauliflower mosaic virus and Cowpea mosaic virus are known to move as virus-like particles through tubules that pass through plasmodesmata into neighboring cells. These tubules are composed of MP (40, 49). Nepo-, Tospo-, and Fabaviruses are also thought to move similarly with the tubulemediated mechanism. There are viruses that are considered to move as a nucleoprotein complex different from virus particles despite their requirement of viral CP for cell-to-cell movement. Cucumber mosaic virus (CMV) is competent to induce tubules protruding from infected protoplasts like the viruses that move as virus-like particles (6). Nevertheless, no such tubules have been found in planta by electron microscopy (3), and mutants incapable of virion formation move successfully from cell to cell (22,44,46). CMV is the type member of the genus Cucumovirus and is one of the most common plant viruses of substantial agricultural significance. CMV infects more than 1,000 species of plants, shrubs, and trees and both monocots and dicots (41). The genomic RNAs of CMV are designated as RNAs 1, 2, and 3, by diminishing size (39). All the RNAs have a cap structure at the 5Ј terminus. The 3Ј portion of all the RNAs is ...

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88, which are translated in a cap-independent manner. The 3' untranslated region of RNA1 contains RNA elements essential for cap-independent translation and negative-strand RNA synthesis. In this study, we investigated how p27 and p88 were engaged in the replication of RCNMV genomic RNAs by using DNA vectors or in vitro transcribed RNAs in protoplasts and in a cell-free extract of evacuolated BY-2 protoplasts. Our results show a cis-preferential requirement of p88, but not of p27, for the replication of RNA1. This mechanism might help to facilitate a switch in the role of RNA1 from mRNA to a replication template.

No abstract

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity. MATERIALS AND METHODS Plants. C. quinoa and tobacco (Nicotiana tabacum L. cv. Xanthi nc) plants were grown in commercially available soil mixtures in a plant growth room at 24 Ϯ 1ЊC with illumination for 16 h per day. cDNA clones. Plasmids pBTF1, pBTF2, and pBTF3W contain the full-length cDNAs of RNAs 1, 2, and 3 of the wild-type BMV, respectively, from which infectious transcripts can be produced by using T7 RNA polymerase (32-34). In vitro transcripts from these plasmids and their progenies will be referred to as the BMV KU2 strain. Plasmids pCY1-T7 and pCY2-T7 (generous gifts from S. Kuwata and M. Suzuki, Japan Tobacco Incorporated) contain the full-length cDNAs of CMV strain Y RNAs 1 and 2, respectively, and infectious viral RNA transcripts can be transcribed in vitro by T7 RNA polymerase from these plasmids (44). A full-length RNA 3 cDNA clone of the wild-type CMV (an isolate of the Y strain) was constructed by joining partial cDNA fragments from pCY32C1 and pCY78 (generous gifts from M. Nakayama, Takeda Chemical Industries, Ltd.). The full-length RNA 3 cDNA fragment was inserted into pUCT7 (34) to

Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.