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Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.

Viral RNAs are specifically selected for packaging during viral infection. Specific packaging occurs through an interaction between viral RNAs and structural coat proteins (CPs). The specific recognition of viral RNAs by CPs plays a crucial role in diverse facets of the viral life cycle and in the packaging event. The binding of CP to specific RNA elements is required for viral protein translation during infection initiation in alfalfa mosaic virus (28) and for the regulation of translation and the initiation of RNA packaging in the RNA phages (38). In retroviruses, nucleocapsid protein is thought to stimulate genomic RNA dimerization, which is required for efficient RNA packaging, reverse transcription, and recombination (14, 31). Many plant viruses require RNA packaging for systemic spread and for cell-to-cell movement (3, 10). Therefore, the CP-RNA interaction is a critical event in the viral life cycle. Compared with the characterization of trans-acting factors such as CP in RNA packaging, however, the RNA elements involved in specific packaging have not been well characterized for many viruses. One of the best-characterized RNA elements is “origin of assembly” of tobacco mosaic virus (36, 40). cis-acting sequences for RNA packaging have also been demonstrated in several icosahedral viruses, including flock house virus (39), Sindbis virus (37), hepatitis B virus (17), the human immunodeficiency viruses (2, 14, 22), and turnip crinkle virus (32). Brome mosaic virus (BMV) is an icosahedral plant RNA virus and is the type member of the genus Bromovirus in the family Bromoviridae in the alphavirus-like superfamily (18). The genome of BMV consists of three species of messenger sense single-stranded RNA (1). RNA1 (3.2 kb) and RNA2 (2.9 kb), which encode the 1a and 2a replicase proteins, respectively (1, 13, 19), are packaged separately into individual particles (21). RNA3 (2.1 kb), which encodes the 3a cell-to-cell movement protein (35), is packaged into a single particle together with subgenomic RNA4 (0.9 kb) (21). RNA4 is synthesized from the minus strand of RNA3 (25) and encodes CP. CP is required for packaging, cell-to-cell movement, and the systemic spread of the virus (29, 33, 34). A highly conserved N-terminal arginine-rich motif in BMV CP plays an important role in BMV RNA packaging through RNA-CP interactions (4, 5, 33, 34). The crystallographic structure of BMV virions has been determined (23). RNA regions or elements involved in the packaging of BMV RNAs have been assigned to the coding region of BMV RNA1 by UV cross-linking and band-shift assays (11) as well as to the 3′-proximal region of the 3a open reading frame (ORF) in RNA3 (9). The tRNA-like structures (TLS) in the 3′-untranslated regions of BMV RNAs also play a crucial role in BMV RNA packaging in vitro (6). In the present study, we delimit a nucleotide sequence required for the efficient packaging of BMV RNA3 and show that 69 nucleotides (nt) in the 3′-proximal region of the BMV 3a ORF, especially a predicted stem-loop structure (30 nt), is essential for the efficient packaging of BMV RNA3. We also propose the presence of elements in the BMV 3a ORF that are involved in the regulation of the copackaging of RNA3 and RNA4.

Spring beauty latent virus (SBLV), a bromovirus, systemically and efficiently infected Arabidopsis thaliana, whereas the well-studied bromoviruses brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) did not infect and poorly infected A. thaliana, respectively. We constructed biologically active cDNA clones of SBLV genomic RNAs and determined their complete nucleotide sequences. Interestingly, SBLV RNA3 contains both the box B motif in the intercistronic region, as does BMV, and the subgenomic promoter-like sequence in the 5' noncoding region, as does CCMV. Sequence comparisons of SBLV, BMV, CCMV, and broad bean mottle virus demonstrated that SBLV is closely related to BMV and CCMV.

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process., Author Summary Intercellular movement of plant viruses is the crucial step during systemic viral infections. Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus, forms movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum in infected cells, which are required for efficient intercellular movement of the virus. We provide evidence that these cortical punctate structures constitute the viral replication complex (VRC), which forms during the early stage of virus infection. Furthermore, we show that a host protein of Nicotiana benthamiana, glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), possibly intercalates between the cortical VRC and MP. Knockdown of NbGAPDH-A diffused subcellular localization of MP and reduced intercellular movement of the virus. Chloroplastic NbGAPDH-A relocalized to the cortical VRC after infection with the virus. Our results suggest that the cortical VRC serves not only as the replication factory of viral RNA but also as a transportation hub, which transports viral RNA to neighboring uninfected cells via plasmodesmata.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Summary We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed ‘mRNA amplification system’ where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 × 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 × 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 × 2FR plant without the treatment and those of the mRNA for IFN were 30–230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 × 2FR plant. The advantages and potential uses of this system are also discussed.

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus , a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser 118 , Glu 132 , Glu 138 , Gln 178 , and Ser 180 ). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Cucumber mosaic cucumovirus (CMV) and brome mosaic bromovirus (BMV) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (CP) for cell-to-cell movement. We have shown that a chimeric BMV with the CMV 3a movement protein (MP) gene instead of its own cannot move from cell to cell in Chenopodium quinoa, a common permissive host for both BMV and CMV. Another chimeric BMV was constructed by replacing both MP and CP genes of BMV with those of CMV (MP/CP-chimera) and tested for its infectivity in C. quinoa, to determine whether the CMV CP has some functions required for the CMV MP-mediated cell-to-cell movement and to exhibit functional difference between CPs of BMV and CMV. Cell-to-cell movement of the MP/CP-chimera occurred, and small local lesions were induced on the inoculated leaves. A frameshift mutation introduced in the CMV CP gene of the MP/CP-chimera resulted in a lack of cell-to-cell movement of the chimeric virus. These results indicate that the viral movement mediated by the CMV MP requires its cognate CP. Deletion of the amino-terminal region in CMV CP, which is not obligatory for CMV movement, also abolished cell-to-cell movement of the MP/CP-chimera. This may suggest some differences in cell-to-cell movement of the MP/CP-chimera and CMV. On the other hand, the sole replacement of BMV CP gene with that of CMV abolished viral cell-to-cell movement, suggesting a possibility that the viral movement mediated by the BMV MP may also require its cognate CP. Functional compatibility between MP and CP in viral cell-to-cell movement is discussed.

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.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes, but distinct host ranges: BMV systemically infects the monocot barley, while CCMV systemically infects the dicot cowpea. We have previously shown that in approximately 10% of inoculated cowpea plants, a CCMV hybrid [CCMV(B3a)] with the 3a cell-to-cell movement protein gene replaced by that of cowpea-nonadapted BMV directs systemic infections, which are caused by secondary mutation(s) of the hybrid virus. Here, to further analyze the role of RNA3 in adaptation to a new host, RNA3 cDNA clones were constructed from total RNA recovered from the uninoculated upper leaves of systemically infected cowpea plants inoculated with CCMV(B3a). Sequence and mutational analysis of two such RNA3 clones revealed that a single codon change (A776 → C) in a conserved motif of the 3a movement protein gene conferred compatibility for systemic infection of a new host, cowpea, suggesting that this site in the 3a gene is directly or indirectly involved in crucial host interactions associated with host-range specificity. The adaptive hybrid viruses carrying this mutation induced exacerbated symptoms, while wt CCMV appeared nearly symptomless, showing that the bromovirus 3a movement protein gene can significantly contribute to regulating symptom development. However, introducing this cowpea-adaptive mutation into the BMV genome had little effect on the ability of BMV to systemically infect barley.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in non-transgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3′ 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 and V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

The nonstructural 3a protein of the positive-strand RNA bromovinuses is required for infection spread in plants and is acrucial determinant of host specificity in systemic infection. To determine the paths of wild-type (wt) bromovirus infection spread, the step at which 3a mutants are arrested, and the nature of the host specificity associated with the 3a gene, we used in situ hybridization to examine infection spread by cowpea chlorotic mottle bromovirus (CCMV) and its derivatives at the level of individual cells in cowpea leaf epidermis. From 1 to 3 days post inoculation (dpi), wt CCMV spread from initially infected cells to adjacent cells, creating expanding infection foci whose radii grew by one additional epidermal cell diameter every 5 hr. By 3 to 4 dpi, vascular elements contacting such foci acted as conduits for further infection spread. By contrast, a 3a frameshift derivative multiplied in initially infected epidermal cells but failed to move into neighboring cells even by 4 dpi, showing that the 3a gene is essential for cell-to-cell spread. Most interestingly, a CCMV derivative with the 3a gene replaced by that of a bromovirus not adapted to cowpea, brome mosaic virus (BMV), initially spread from cell to cell in cowpea plants, but stopped spreading between 1 and 2 dpi, when most infection foci encompassed 40–80 epidermal cells. Thus, the host-specificity restriction imposed by BMV 3a protein did not result from an inability to direct the spread of infection out of initially infected cowpea cells, but from a much later block. The apparent absence of any preexisting anatomical boundary at the limit of infection spread and localized tissue changes at the infection foci suggested that induced host responses might have contributed to this block.

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.

Positive-strand RNA viruses require host intracellular membranes for replicating their genomic RNAs. In this study, we determined the domains and critical amino acids in p27 of Red clover necrotic mosaic virus (RCNMV) required for its association with and targeting of ER membranes in Nicotiana benthamiana plants using a C-terminally GFP-fused and biologically functional p27. Confocal microscopy and membrane-flotation assays using an Agrobacterium-mediated expression system showed that a stretch of 20 amino acids in the N-terminal region of p27 is essential for the association of p27 with membranes. We identified the amino acids in this domain required for the association of p27 with membranes using alanine-scanning mutagenesis. We also found that this domain contains amino acids not critical for the membrane association but required for the formation of viral RNA replication complexes and negative-strand RNA synthesis. Our results extend our understanding of the multifunctional role of p27 in RCNMV replication.

The complete nucleotide sequence of a set of cDNA clones designated KU1 strain, from which infectious in vitro transcripts can be transcribed, of the ATCC66 strain of brome mosaic virus (BMV) was determined and compared with the Russian strain of BMV in both nucleotide and the deduced amino acid sequence. Each corresponding genomic RNA in the KU1 strain and the Russian strain was homologous by more than 97% in nucleotide sequence. Base substitution of the transition type was more frequent than that of the transversion type in all three genomic RNAs. Amino acid changes in three regions where significant amino acid sequence homology is shown among the Sindbis-like plant RNA viruses were mostly substitutions for amino acids categorized to similar groups. The nucleotide sequence of the 5′ noncoding region in RNA1 and RNA3 was identical in the corresponding RNAs of the two strains while seven base changes were observed in RNA2. Any base change in the 3′ noncoding region did not disrupt a predicted secondary structure. Two adjacent adenine residues were absent in the noncoding leader sequence of the coat protein gene of both KU1 strain and ATCC66 strain. The intercistronic oligo(A) tract in RNA3 of the ATCC66 strain was heterogeneous in length in which more than two guanine residues were observed by direct RNA sequencing, while one guanine residue was present in the RNA3 of the cDNA clone of the KU1 strain.

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3′ untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans . The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.

We introduced replicase genes of brome mosaic virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3' non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3.

Full-length biologically active cDNAs of brome mosaic virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5' ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower mosaic virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5' end were infectious but RNAs transcribed from cDNAs with similar sequences were not.

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 5′→3′ 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 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae .

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the α chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

The 3a movement protein (B3a) of brome mosaic virus (BMV) plays essential roles in the cell-to-cell movement of BMV. B3a is known to bind nucleic acids, to transport RNA to neighbouring cells, and to form tubular structures. Here, we tested the assumption that phosphorylation may be a mechanism that regulates B3a functions and showed that not only B3a but also the coat protein, BCP, was phosphorylated in BMV-infected barley protoplasts. Both BCP and B3a were detected in a complex immunoprecipitated from BMV-infected protoplasts with anti-B3a antiserum, implying an interaction between BCP and B3a.

RNA interference (RNAi) is a sequence-specific gene regulatory mechanism conserved among diverse eukaryotes. The sequence specificity in RNAi is determined by a family of 18- to 30-nucleotide (nt) regulatory small RNAs (for review, see Aravin and Tuschl 2005). Two major classes of endogenous small RNAs have been characterized: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs—the best-characterized endogenous small RNAs in eukaryotes—have been identified in diverse plants and animals, and are mainly involved in development and differentiation. miRNAs are processed from miRNA precursors (pre-miRNAs) with a stem-loop structure and regulate gene expression through translational repression or mRNA cleavage (for reviews, see Ambros 2004; Bartel 2004; He and Hannon 2004; Du and Zamore 2005). siRNAs are generated from long double-stranded RNA (dsRNA) and are mainly involved in defense against molecular parasites including viruses, transposons, and transgenes through RNAi (Sijen and Plasterk 2003; Shi et al. 2004). Endogenous siRNAs have been classified into at least three subclasses: repeat-associated siRNAs (rasiRNAs), trans-acting siRNAs (ta-siRNAs), and siRNAs derived from natural antisense transcripts (nat-siRNAs) (Lippman and Martienssen 2004; Peragine et al. 2004; Borsani et al. 2005). rasiRNAs corresponding to repetitive elements repress the repeat sequences at the transcriptional or post-transcriptional level and maintain a centromeric heterochromatic structure (Lippman and Martienssen 2004). rasiRNAs have been cloned and sequenced in Schizosaccharomyces pombe, Trypanosoma brucei, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Arabidopsis thaliana, but not in mammals (for review, see Aravin and Tuschl 2005). In D. melanogaster and D. rerio, however, the lengths of rasiRNAs are longer than those of miRNAs (Ambros et al. 2003; Chen et al. 2005). ta-siRNAs are processed from dsRNAs synthesized by an endogenous RNA-dependent RNA polymerase (RdRp) utilizing endogenous mRNAs as template (Peragine et al. 2004). nat-siRNAs are processed from dsRNAs formed between endogenous sense and antisense transcripts (Borsani et al. 2005). In addition to miRNA and siRNA, several classes of small RNAs have been reported. scanRNAs (scnRNAs; 27–30 nt) in Tetrahymena thermophila are thought to be derived from dsRNA precursors and guide DNA elimination (Mochizuki et al. 2002; Lee and Collins 2006). small RNAs (23–24 nt) in T. thermophila are mapped to the genome in clusters and are oriented in the same direction (Lee and Collins 2006). X-cluster small RNAs in C. elegans are derived from a locus on Chromosome X extending ∼2 kb and are oriented in the same direction (Ambros et al. 2003). Several factors including DCR-1, Dicer-related helicase DRH-1, the RdRp RRF-1, and the exonuclease ERI-1 are reported to be required for the accumulation of X-cluster small RNAs (Duchaine et al. 2006). The roles of 23- to 24-nt small RNAs in T. thermophila and X-cluster small RNAs in C. elegans remain unknown. Key factors required for the biogenesis and function of these small RNAs are Dicer and Argonaute proteins (for review, see Tomari and Zamore 2005). Dicer is an RNaseIII-like enzyme that recognizes dsRNAs, including pre-miRNAs, and processes them into double-stranded small RNAs (Hutvagner et al. 2001). The Dicer-generated double-stranded small RNAs are recruited by Argonaute, and then a strand (called the passenger strand) is released from Argonaute and the other strand (called the guide strand) remains associated with Argonaute as a guide to regulate gene expression (Matranga et al. 2005). Based on the amino acid sequence alignments, the Argonaute protein family has been subdivided into two subfamilies, referred to as the Argonaute and Piwi families (Carmell et al. 2002). Mice have four Argonaute family genes (AGO1–4) and three Piwi family genes (Miwi, Mili, and Piwil4). AGO1–4, which are ubiquitously expressed in many tissues (Lu et al. 2005), recruit miRNAs (Liu et al. 2004). Piwi family genes are expressed predominantly in male germline cells (Kuramochi-Miyagawa et al. 2001) and have crucial roles in spermatogenesis. Disruption of Miwi causes spermatogenic arrest at the beginning of the round spermatid stage (Deng and Lin 2002). Spermatogenesis in Mili-null mice is blocked completely at the early prophase of the first meiosis (Kuramochi-Miyagawa et al. 2004). The molecular functions and associated small RNAs of Piwi family proteins remain unknown. Whether endogenous siRNAs are present in mouse is unclear for three reasons: (1) There has been no report of siRNA cloning in mammals; (2) there is no evidence for the presence of RdRp activity in mammals, which generates dsRNAs, namely the precursors of siRNAs; and (3) induction of the interferon pathway by dsRNAs usually results in cell death, suggesting that mammalian cells may not tolerate dsRNAs (Elbashir et al. 2001a). However, in mouse oocytes and preimplantation embryos the interferon response is suppressed, and injection of long dsRNAs results in specific reduction in the amount of target mRNAs (Svoboda et al. 2000; Yan et al. 2005). Furthermore, retrotransposons and their antisense transcripts are expressed in mouse preimplantation embryos (Peaston et al. 2004; Svoboda et al. 2004a), and knockdown of Dicer in mouse preimplantation embryos results in a 50% increase in IAP and MuERV-L retrotransposons (Svoboda et al. 2004a). These studies suggest that endogenous dsRNA-induced RNAi can occur in mouse oocytes and early embryos. In mammals, hundreds of miRNAs have been identified by extensive small RNA cloning and bioinformatic analyses (Lagos-Quintana et al. 2001; Houbaviy et al. 2003; Lim et al. 2003). However, other classes of small RNAs have not been studied. In this study, as a step to obtain a whole picture of the small RNA population in mammals, we have cloned and sequenced small RNAs from oocytes and testes. We identified two classes of small RNAs other than miRNAs. One class comprised ∼22-nt retrotransposon-derived siRNAs in oocytes, which showed characteristics of small RNAs associated with RNAi. The other class comprised novel 26- to 30-nt germline small RNAs (gsRNAs) that were present in male germ cells and had some interesting features that were distinct from those of siRNAs and miRNAs. The features of the two classes of small RNAs suggest their distinct origins and functions and the existence of two separate small RNA pathways in the mouse germline.

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200–300 nm in length. SDS–PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.Key words: Saprospira, gliding motility, Saprospira cytoplasmic fibril protein (SCFP).

Thraustochytrids are marine fungoid protists classified in the class Labyrinthulea in the kingdom Chromista (8, 9). They are comprised of six genera (33, 47), Althornia (26), Aplanochytrium (2), Japonochytrium (32), Schizochytrium (18), Thraustochytrium (59), and Ulkenia (13). However, it has been shown that the current classification of these genera based on morphology does not agree with the molecular phylogenetic relationships based on the 18S rRNA gene sequences (21). Currently, in order to resolve the confusion regarding the classification and nomenclature of the thraustochytrids, further comparative studies based on morphology, molecular phylogeny, and chemotaxonomy are under way (R. Yokoyama, personal communication). Hence, some of the thraustochytrid strains tested in the present study have not been fully identified yet (Table ​(Table11). TABLE 1. Infection specificities of SssRNAV with 19 strains of marine microorganisms Thraustochytrids are distributed in saline lakes and in marine, estuarine, and deep-sea waters throughout the world (35, 47, 52), and they have been isolated from algal and plant material, as well as from sediments and water (14, 37, 55, 59). Recently, a rapid direct detection technique for thraustochytrids using the fluorogenic acriflavine dye was developed (53). Using this method, Naganuma et al. (39) estimated the abundance of thraustochytrids in the Seto Inland Sea of Japan and demonstrated that the biovolume of thraustochytrids in coastal waters could reach 43% of the bacterial biovolume. The wide distribution and high abundance of these organisms indicate their ecological importance as decomposers (39, 52). In addition, thraustochytrids are known to produce large amounts of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid and docosapentaenoic acid (44), which are considered important food resources for higher organisms in marine systems (30, 34, 50). Furthermore, some species of thraustochytrids are known to be pathogens of mollusks, such as octopuses and bivalves (1, 46, 49). Because of these distinctive features, the ecological significance of thraustochytrids in the coastal ecosystems has been highlighted (50, 51). On the other hand, viruses are very abundant in marine environments (3, 48). Viruses and virus-like particles (VLPs) have been discovered in a variety of phytoplankton and bacteria (48, 54, 64) and have been recognized as important agents in controlling bacterial and algal biomass (4, 41, 48) and nutrient cycling (17, 66) and in maintaining the biodiversity of bacteria and microalgae (5, 10, 12). So far, more than 13 viruses infecting marine microalgae have been isolated and characterized (5). The majority of these viruses are large (100- to 200-nm) double-stranded DNA viruses, and they are either included in the family Phycodnaviridae (5, 58, 65) or considered most likely to belong to this family based on some characteristics (7, 16, 23, 43, 57, 62). In contrast, there have been only a small number of reports describing RNA viruses infecting marine eukaryotic microorganisms. So far, three RNA viruses infecting marine eukaryotic microalgae have been isolated; two of them are single-stranded RNA (ssRNA) viruses (HaRNAV and HcRNAV), and one is a double-stranded RNA (dsRNA) virus (MpRNAV). HaRNAV is infectious to one of the most noxious bloom-forming phytoflagellates, Heterosigma akashiwo (Raphidophyceae), and has an ssRNA genome that is 9.1 kb long (61). HcRNAV is infectious to the bivalve-killing dinoflagellate Heterocapsa circularisquama and has an approximately 4.4-kb ssRNA genome (63). MpRNAV is infectious to the cosmopolitan phytoplankter Micromonas pusilla and harbors 11 segments of dsRNA as the viral genome, the total length of which is 25.5 kbp (6). In addition, the relationship between protists and their viruses is poorly understood. Nagasaki et al. (40, 42) observed VLPs in marine apochlorotic flagellates and suggested that viral infection might be one of the factors affecting their dynamics. Garza and Suttle (15) isolated and characterized a large dsDNA virus infecting a marine heterotrophic nanoflagellate, Bodo sp., which also shared some characteristics with viruses belonging to the family Phycodnaviridae. For thraustochytrids, Kazama and Schornstein (28, 29) found herpes-type VLPs in Thraustochytrium sp. (Thraustochytriaceae, Labyrinthulea) which were roundish, enveloped, 110 nm in diameter, and predicted to have a DNA genome. However, because the VLPs were not successfully brought into culture, further study could not be completed. In the present report, we describe the isolation and characterization of a novel ssRNA virus infecting Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea). To our knowledge, this is the first report describing the biological properties of an RNA virus infecting marine fungoid protists.

RNA interference (RNAi) is a post-transcriptional gene-regulatory 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.

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 movement protein (MP) of Brome mosaic virus (BMV) depends on the coat protein (CP) to mediate the cell-to-cell movement of BMV and CCMV(B3a), a recombinant Cowpea chlorotic mottle virus (CCMV) expressing BMV MP. Previous studies identified gain-of-function mutations in the central region of BMV MP that enable CCMV(B3a) to adapt to a resistant host. This study demonstrates that all adaptation-related MPs can partially or almost fully mediate the cell-to-cell movement of CCMV(B3a) and BMV without CP. Based on these results, we discuss adaptation mechanisms of CCMV(B3a) and the role of the central region of MP in the determination of virus movement mode.

A hybrid Cowpea chlorotic mottle virus (CCMV) with the movement protein (MP) gene replaced with that of the closely related Brome mosaic virus cannot infect cowpea systemically. Twenty-nine spontaneous mutants from the hybrid CCMV capable of systemic infection in cowpea appeared through biased codon changes that resulted in Lys or Arg at five specific positions in the MP gene. In this study, we report that systemic infection of cowpea with the hybrid CCMV can be achieved by artificial codon changes that do not result in Lys or Arg. We discuss mechanisms that restrict the occurrence of cowpeaadapted mutants in nature.

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 (3′TE-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 3′TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.

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.

Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, and is closely related to Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV). Compatible interactions between viral components are required for successful infection of plants by BMV and CCMV. To further our understanding of interactions between bromovirus components, we used SBLV to produce reassortants among the three bromoviruses. We found that SBLV RNA 2 functioned with heterologous bromovirus RNA 1 in infections of whole plants and protoplasts of Nicotiana benthamiana, although SBLV RNA 1 did not function with heterologous bromovirus RNA 2. A DNA-based transient assay for 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively further suggested that SBLV 2a protein may function in combination with heterologous bromovirus 1a protein. Moreover, analysis of the ability of reassortants to spread locally revealed that an RNA 2-mediated interaction between viral components may be required for efficient cell-to-cell movement of bromoviruses.

Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants. This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components. To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 × 106independent clones, using a yeast two-hybrid system. Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated. One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1. HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues. A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1. Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley. These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.

Brome mosaic virus (BMV) requires the coat protein (CP) for cell-to-cell movement whereas Cowpea chlorotic mottle virus (CCMV), from the same genus, does not. Chimeric viruses created by exchanging the movement protein (MP) gene between the viruses can move from cell to cell. We show that interference in CP expression impaired the movement of the chimeric CCMV with the BMV MP gene but not of the chimeric BMV with the CCMV MP gene. We thus conclude that the MP gene plays a crucial role in determination of the virus-specific CP requirement in bromovirus cell-to-cell movement.

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 fluorescent protein-based 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 first RNA silencing suppressor identified in negative-strand RNA viruses.

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5′ cap. Both RNAs were largely resistant to 3′ polyadenylation and ligation, suggesting the presence of an interfering 3′ structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5′ and 3′ non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT–PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3′ terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0·4 kb) that was newly synthesized and corresponded to the 3′-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vas-culature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Nous avons construit une genotheque d'ADNc egalise (normalise) a partir du champignon Colletotrichum lagenarium dans le but de mettre au point un systeme efficace de criblage des genes exprimes differemment chez ce champignon. Des conidies en processus de differentiation en hyphes applatis (appressoriums) ont servi de materiel de base dans la construction de cette genotheque d'ADNc pour l'isolement des genes controlant les etapes de developpement reliees au processus infectieux des conidies. La normalisation des ADNc a ete faite deux fois par une methode cinetique et les produits ont ete clones dans un vecteur plasmidique. Une hybridation de colonies avec neuf sondes de differentes abondances a revele une diminution de la variation de l'abondance d'au moins 276 fois dans la genotheque originale a 10 fois dans la genotheque d'ADNc normalise, confirmant ainsi que l'ADNc etait egalise avec succes. Une hybridation differentielle de 1900 clones d'ADNc dans la banque d'ADNc normalise et une analyse d'empreintes d'ARN de clones candidats nous a permis d'identifier 11 clones d'ADNc independants identifies CADI a CAD11 qui etaient exprimes chez les hyphes se differenciant en appressoriums mais pas chez les hyphes vegetatifs. Les copies de CADI et CAD2 s'accumulaient fortement dans les conidies preincubees tandis que les copies de CAD3 et de CAD4 s'accumulaient en grand et en petit nombre respectivement. Les quatre copies CAD augmentaient en nombre durant les premieres etapes du processus de formation des appressoriums. L'analyse de la sequence de CADI suggere que le gene CADI coderait un polypeptide de 101 acides amines presentant une homologie avec les metallothioneines. Selon la sequence obtenue, le gene CAD2 coderait un polypeptide de 278 acides amines et il presente une forte homologie avec certains des genes codant l'aflatoxine chez Aspergillus parasiticus et la sterigmatocystine chez A. nidulans.

Cowpea chlorotic mottle bromovirus (CCMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The 3a protein was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that the CCMV 3a protein (CC3a) bound single-stranded (ss) RNA cooperatively without sequence specificity. Binding competition analysis showed that CC3a bound ss-nucleic acids more strongly than double-stranded nucleic acids.

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein; the function of which is as yet unknown. This protein has non-sequence-specific DNA binding activity and is associated with viral particles, suggesting that the ORF III product (P3) is involved in the folding of CaMV DNA during encapsidation. In this study, we demonstrated that P3 forms a tetramer in CaMV-infected plants. A P3-related protein with an apparent molecular weight of 60 kDa was detected by Western blotting analysis using anti-P3 antiserum under non-reducing conditions, while only 15 kDa P3 was detected under reducing conditions. Analysis of P3 using viable mutants with a 27-bp insertion in either ORF III or IV revealed that the 60 kDa protein was a tetramer of P3. The P3 tetramer co-sedimented with viral coat protein in multiple fractions on sucrose gradient centrifugation, suggesting that P3 tetramer binds to mature and immature virions. These results strongly suggested that CaMV P3 forms a tetramer in planta and that disulfide bonds are involved in its formation and/or stabilization. The finding of P3 tetramer in planta suggested that viral DNA would be folded compactly by the interaction with multiple P3 molecules, which would form tetramers, while being packaged into the capsid shell.

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.