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.

Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3 ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5 cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5 cap and 3 poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5 cap-and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3 untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5 cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3 UTR, designated the 3 translation element of Dianthovirus RNA1 (3TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and Luteovirus. A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.The genomic RNA of a positive-sense single-stranded RNA virus serves two essential functions at the start of the viral replication cycle in infected cells: as a template for negative strands and as mRNA for viral proteins. Virtually all eukaryotic cellular mRNAs are capped and polyadenylated. The 5Ј cap structure (m 7 GpppN) serves as the binding site for eukaryotic initiation factor 4F (eIF4F), composed of eIF4G, eIF4E, and eIF4A, which assists the binding of 40S ribosomes to mRNAs (15). The poly(A) tail serves as the binding site for the poly(A)-binding protein, which stabilizes eIF4F binding to the 5Ј cap (43). Consequently, the 5Ј cap and 3Ј poly(A) tail are critical in recruiting translational machinery for the efficient translation of encoded proteins. However, a variety of eukaryotic viral mRNAs lack the 5Ј cap and/or poly(A) tail and have therefore developed alternative strategies for translation regulation.For Tobacco mosaic virus, which has a capped genome and which lacks the poly(A) tail, the pseudoknot domain in the 3Ј untranslated region (UTR) appears to substitute functionally for the poly(A) tail and enhances translation (27). In addition, the poly(CAA) region in the 5Ј UTR (termed ⍀) is responsible for translation enhancement (9). The genomic RNA of picornaviruses has a poly(A) tail but lacks the 5Ј cap. In this case, the internal ribosome entry site (IRES) in the 5Ј UTR recruits ribosomes directly and enables cap-independent translation initiation to occur (21). In contrast to the IRESs of pi...

Heterocapsa circularisquama RNA virus (HcRNAV) has at least two ecotypes (types UA and CY) that have intraspecies host specificities which are complementary to each other. We determined the complete genomic RNA sequence of two typical HcRNAV strains, HcRNAV34 and HcRNAV109, one of each ecotype. The nucleotide sequences of the viruses were 97.0% similar, and each had two open reading frames (ORFs), ORF-1 coding for a putative polyprotein having protease and RNA-dependent RNA polymerase (RdRp) domains and ORF-2 encoding a single major capsid protein. Phylogenetic analysis of the RdRp amino acid sequence suggested that HcRNAV belongs to a new previously unrecognized virus group. Four regions in ORF-2 had amino acid substitutions when HcRNAV34 was compared to HcRNAV109. We used a reverse transcriptionnested PCR system to amplify the corresponding regions and also examined RNAs purified from six other HcRNAV strains with known host ranges. We also looked at natural marine sediment samples. Phylogenetic dendrograms for the amplicons correlated with the intraspecies host specificities of the test virus strains. The cloned sequences found in sediment also exhibited considerable similarities to either the UA-type or CY-type sequence. The tertiary structure of the capsid proteins predicted using computer modeling indicated that many of the amino acid substitutions were located in regions on the outside of the viral capsid proteins. This strongly suggests that the intraspecies host specificity of HcRNAV is determined by nanostructures on the virus surface that may affect binding to suitable host cells. Our study shows that capsid alterations can change the phytoplankton-virus (host-parasite) interactions in marine systems.Only five RNA viruses are known to infect marine eukaryotic microorganisms. The following four viruses are singlestranded RNA (ssRNA) viruses: Heterosigma akashiwo RNA virus (HaRNAV) infects the noxious bloom-forming raphidophyte Heterosigma akashiwo (Raphidophyceae) (26); Rhizosolenia setigera RNA virus infects the bloom-forming diatom Rhizosolenia setigera (20); Heterocapsa circularisquama RNA virus (HcRNAV) infects the bivalve-killing bloom-forming dinoflagellate Heterocapsa circularisquama (30); and Schizochytrium sp. single-stranded RNA virus (SssRNAV) infects the marine fungoid protist Schizochytrium sp. (Labyrinthulae, Thraustochytriaceae) (27). One of the five viruses is a doublestranded RNA (dsRNA) virus (Micromonas pusilla RNA virus) that infects the cosmopolitan phytoplankton Micromonas pusilla (1). Detailed genomic analysis has been performed for two of these viruses, HaRNAV (18) and SssRNAV (Takao et al., unpublished data). Hence, genomic studies of the three other marine RNA viruses are required. In this paper we describe the genome of HcRNAV, which infects H. circularisquama.HcRNAV infection is strain specific rather than species specific because about 6,000 combinations of cross-infection tests between H. circularisquama strains and HcRNAV strains showed that HcRNAV strains are divided rou...

The pepper L gene conditions the plant's resistance to Tobamovirus spp. Alleles L(1), L(2), L(3), and L(4) confer a broadening spectra of resistance to different virus pathotypes. In this study, we report the genetic basis for the hierarchical interaction between L genes and Tobamovirus pathotypes. We cloned L(3) using map-based methods, and L(1), L(1a), L(1c), L(2), L(2b), and L(4) using a homology-based method. L gene alleles encode coiled-coil, nucleotide-binding, leucine-rich repeat (LRR)-type resistance proteins with the ability to induce resistance response to the viral coat protein (CP) avirulence effectors by themselves. Their different recognition spectra in original pepper species were reproduced in an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana. Chimera analysis with L(1), which showed the narrowest recognition spectrum, indicates that the broader recognition spectra conferred by L(2), L(2b), L(3), and L(4) require different subregions of the LRR domain. We identified a critical amino acid residue for the determination of recognition spectra but other regions also influenced the L genes' resistance spectra. The results suggest that the hierarchical interactions between L genes and Tobamovirus spp. are determined by the interaction of multiple subregions of the LRR domain of L proteins with different viral CP themselves or some protein complexes including them.

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3'TE-DR1) in the 3' untranslated region (UTR). In this study, we analyzed the roles of 5' and 3' UTRs of RNA1 in 3'TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (Luc) reporter assay system. Most mutations introduced into RNA1 5' UTR in reporter Luc mRNA abolished or greatly reduced cap-independent translation in BY-2 protoplasts, whereas those mutations had no or much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stem-loop structure predicted in the 5' proximal region of RNA1 plays important roles in both translation and RNA stability. We also show that 3'TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts.

No abstract

The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.The genomic RNAs of positive-strand RNA viruses play multiple roles during the infection cycle. Upon entering host cells, they act as mRNAs that direct viral protein synthesis; they serve as templates for genomic RNA replication; they are packaged into progeny virions; and in some viruses they serve as templates for subgenomic RNA synthesis and act as regulators of gene expression. In these processes, viral RNAs function as cis-acting elements that recruit translation factors, RNA replicase component proteins, and structural proteins (1, 5, 6). Viruses achieve infection by properly regulating these processes, because these processes sometimes conflict with one another (2,7,26).To investigate the RNA replication and gene expression mechanisms of RNA viruses, we used Red clover necrotic mosaic virus (RCNMV). RCNMV is classified in the family Tombusviridae and the genus Dianthovirus. The genome of RCNMV is divided into two RNA molecules, RNA1 and RNA2 (10,12,25), unlike the case for other viruses in the Tombusviridae family, which have a monopartite RNA genome. RNA1 has no cap structure at the 5Ј end (22), has no poly(A) tail at the 3Ј end (21, 35), and encodes putative RNA replicase components, a 27-kDa protein (p27), and an 88-kDa protein (p88). p88 has an RNA-dependent RNA polymerase motif (15) and is produced by programmed Ϫ1 ribosomal frameshifting (13, 38). RNA1 also encodes a 37-kDa coat protein (CP) that is expressed from a subgenomic RNA (40). The 3Ј untranslated region (UTR) of RCNMV RNA1 functions as a primary determinant of temperature-sensitive viral RNA accumulation (21) and can function alone without its 5Ј UTR as a cap-independent tr...