Effects of the cucumber mosaic virus 2a protein on aphid-plant interactions in Arabidopsis thaliana.

The cucumber mosaic virus (CMV) 2a RNA-dependent RNA polymerase protein has an additional function in Arabidopsis thaliana, which is to stimulate feeding deterrence (antixenosis) against aphids. Antixenosis is thought to increase the probability that aphids, after acquiring CMV particles from brief probes of an infected plant's epidermal cells, will be discouraged from settling and instead will spread inoculum to neighbouring plants. The amino acid sequences of 2a proteins encoded by a CMV strain that induces antixenosis in A. thaliana (Fny-CMV) and one that does not (LS-CMV) were compared to identify residues that might determine the triggering of antixenosis. These data were used to design reassortant viruses comprising Fny-CMV RNAs 1 and 3, and recombinant CMV RNA 2 molecules encoding chimeric 2a proteins containing sequences derived from LS-CMV and Fny-CMV. Antixenosis induction was detected by measuring the mean relative growth rate and fecundity of aphids (Myzus persicae) confined on infected and on mock-inoculated plants. An amino acid sequence determining antixenosis induction by CMV was found to reside between 2a protein residues 200 and 300. Subsequent mutant analysis delineated this to residue 237. We conjecture that the Fny-CMV 2a protein valine-237 plays some role in 2a protein-induced antixenosis.

Engineering partial resistance to cucumber mosaic virus in tobacco using intrabodies specific for the viral polymerase.

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Heterologous replicase driven 3' end repair of Cucumber mosaic virus satellite RNA.

To investigate the extent of the 3' end repair in a satellite RNA of Cucumber mosaic virus (CMV) strain Q (Q(sat)) by a heterologous Tomato aspermy virus (TAV), a set of biologically active agrotransformants corresponding to the three genomic RNAs of TAV was developed. Analysis of Nicotiana benthamiana plants agroinfiltrated with TAV and either wild type or each of the six 3' deletion mutants of Q(sat) revealed that (i) heterologous replicase failed to generate Q(sat) multimers, a hallmark feature of homologous replicase dependent replication of Qsat; (ii) manifestation of severe symptom phenotypes and progeny analysis suggested that heterologous replicase was competent to repair Q(sat) deletion mutants lacking up to 3'13 nucleotides (nt) but not beyond and (iii) comparative in silico analysis indicated that the 3' secondary structural features of the repaired Q(sat) progeny from heterologous vs homologous driven replicases are remarkably very similar. The significance of these observations is discussed.

Transgenic tobacco lines expressing defective CMV replicase-derived dsRNA are resistant to CMV-O and CMV-Y.

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909-1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.

A tobacco CBL-interacting protein kinase homolog is involved in phosphorylation of the N-terminal domain of the cucumber mosaic virus polymerase 2a protein.

The replication and transcription of cucumber mosaic virus (CMV) are catalyzed by multi-protein complex RNA-dependent RNA polymerase (RdRp), which is composed of the viral-encoded 1a and 2a proteins with host factors. We have reported that the N-terminal region of the polymerase 2a protein, composed of 126 amino acids, is required for interaction with the helicase 1a protein, and that the phosphorylation of the region abrogated interaction with the 1a protein, suggesting a mechanism of resistance in host plants against viral infection. Here, we found that three protein 2a kinases, of 60, 55, and 38 kDa, co-purified with the tobacco membrane fraction in an in-gel kinase assay. By yeast two-hybrid library screening using the N-terminal 126 amino acids of 2a as a bait, we identified CBL-interacting protein kinase 12 (NtCIPK12) corresponding to 55 kDa protein 2a kinase. The bacterially expressed protein kinase showed protein 2a kinase (t2aK) activity in vitro. We found that NtCIPK12 stabilized upon CMV infection at the post-translational level, and accumulated more heavily to the membrane than in the cytosol.

Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene.

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

Environment determines fidelity for an RNA virus replicase.

The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.

Evidence for interaction between the 2a polymerase protein and the 3a movement protein of Cucumber mosaic virus.

The genome of Cucumber mosaic virus consists of three single-stranded RNA molecules, RNAs 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome and have been implicated in movement of the viral RNAs in plants. The 3a movement protein (MP), encoded by RNA 3, is essential for transferring the RNA genomes from infected cells to adjacent cells across the plasmodesmata. Far-Western analysis demonstrated that bacterially expressed 2a polymerase protein directly interacted with the MP. Interaction was confirmed in a yeast two-hybrid assay, and co-immunoprecipitation analysis showed that the MP interacted only with the 2a polymerase protein. A yeast three-hybrid assay showed that the 1a-2a protein interaction relevant for replicase complex formation was not affected by the MP. Although the MP has no affinity for the 1a protein, it interacted indirectly with the 1a protein via the 2a polymerase protein. These results suggest that the replicase complex may be involved in movement through its interaction with the MP.

Cucumber mosaic virus 2a polymerase and 3a movement proteins independently affect both virus movement and the timing of symptom development in zucchini squash.

The basis for differences in the timing of systemic symptom elicitation in zucchini squash between a pepper strain of Cucumber mosaic virus (Pf-CMV) and a cucurbit strain (Fny-CMV) was analysed. The difference in timing of appearance of systemic symptoms was shown to map to both RNA 2 and RNA 3 of Pf-CMV, with pseudorecombinant viruses containing either RNA 2 or RNA 3 from Pf-CMV showing an intermediate rate of systemic symptom development compared with those containing both or neither Pf-CMV RNAs. Symptom phenotype was shown to map to two single-nucleotide changes, both in codons for Ile at aa 267 and 168 (in Fny-CMV RNAs 2 and 3, respectively) to Thr (in Pf-CMV RNAs 2 and 3). The differential rate of symptom development was shown to be due to differences in the rates of cell-to-cell movement in the inoculated cotyledons, as well as differences in the rate of egress of the virus from the inoculated leaves. These data indicate that both the CMV 3a movement protein and the CMV 2a polymerase protein affect the rate of movement of CMV in zucchini squash and that these two proteins function independently of each other in their interactions with the host, facilitating virus movement.

Interaction of replicase components between Cucumber mosaic virus and Peanut stunt virus.

Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) each have genomes consisting of three single-stranded RNA molecules: RNA 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome. Although RNA 3 is exchangeable between CMV and PSV, exchange of RNA 1 and 2 between the two viruses has been unsuccessful. In this study, reassortants containing PSV RNA 1 and CMV RNA 2 together with RNA 3 of CMV or PSV were shown to be able to replicate their genomic RNA, but not to transcribe subgenomic RNA 4 in tobacco protoplasts. Conversely, the reassortant consisting of CMV RNA 1 and PSV RNA 2 together with RNA 3 of CMV or PSV could not replicate. Subsequently, a yeast two-hybrid system was used to analyse the in vivo interaction between the 1a and 2a proteins. The C-terminal half of PSV-1a protein interacted with the N-terminal region of 2a protein of both PSV and CMV, but the C-terminal half of CMV-1a and the N-terminal region of PSV-2a did not interact. These results suggest that RNA replication in the interspecific reassortant between CMV and PSV requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for transcription of subgenomic RNA 4.

Two amino acids on 2a polymerase of Cucumber mosaic virus co-determine hypersensitive response on legumes.

The hypersensitive response (HR) is one of the most important defense responses during the incompatible interaction between plant and pathogen. The viral determinant of HR on legumes induced by Cucumber mosaic virus (CMV) was studied, and our previous results showed that 243 nucleotides on 2a polymerase gene of CMV were involved in the induction of HR on legumes. With further analysis of the nucleotides and amino acids in this region, the amino acids Phe and Ala at positions 631 and 641 in the 2a polymerase of CMV-Fny, a legume local necrotic strain, were specifically exchanged to Tyr and Ser, respectively and simultaneously, in the 2a polymerase of CMV-P1, a legume systemic infecting strain, and three point mutants were constructed. The point mutant Fny-F/Y (Phe631 to Tyr) induced large necrotic lesions instead of pinpoint lesions, and the size of lesions could enlarge from initial sites. The point mutant Fny-A/S (Ala641 to Ser) induced similar symptoms as CMV-Fny. The double-point mutant Fny-FA/YS (Phe631 to Tyr and Ala641 to Ser) infected the legumes systemically without HR. These data indicate that the induction of HR on legumes is co-determined by two amino acids at positions 631 and 641 in CMV 2a polymerase.

Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex.

The 2a (polymerase) protein of cucumber mosaic virus (CMV) was shown to be phosphorylated both in vivo and in vitro. In vitro assays using 2a protein mutants and tobacco protein kinases showed that the 2a protein has at least three phosphorylation sites, one of which is located within the N-terminal 126 amino acid region. This region is essential and sufficient for interaction with the CMV 1a protein. When phosphorylated in vitro, the 2a protein N-terminal region failed to interact with the 1a protein. Since the 1a-2a interaction is essential for the replication of CMV, this suggests that phosphorylation of the N-terminal region of the 2a protein negatively modulates the interaction in vivo, and may have a regulatory role acting directly in viral infection.

Completion of RNA synthesis by viral RNA replicases.

How the 5'-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA-DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5'-terminus and the C2'-hydroxyl of a ribose at the third nucleotide from the 5'-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: implications for RNA-RNA recombination.

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

Efficient and specific initiation of subgenomic RNA synthesis by cucumber mosaic virus replicase in vitro requires an upstream RNA stem-loop.

We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions -28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions -28 to -5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus.

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

Differential roles of the 5' untranslated regions of cucumber mosaic virus RNAs 1, 2, 3 and 4 in translational competition.

RNA species of plant tripartite RNA viruses show distinct translational activities in vitro when the viral RNA concentration is high. However, it is not known what causes the differential translation of virion RNAs. Using an in vitro wheat germ translation system, we investigated the translation efficiencies and competitive activities of chimeric cucumber mosaic virus (CMV) RNAs that contained viral untranslated regions (UTRs) and a luciferase-coding sequence. The chimeric RNAs exhibited distinct translation efficiencies and competitive activities. For example, the translation of chimeric CMV RNA 4 was about 40-fold higher than that of chimeric CMV RNA 3 in a competitive environment. The distinct translation resulted mainly from differences in competitive activities rather than translation efficiencies of the chimeric RNAs. The differential competitive activities were specified by viral 5 UTRs, but not by 3 UTRs or viral proteins. The competitive translational activities of the 5 UTRs were as follows: RNA 4 (coat protein)>RNAs 2 and 1 (2a and 1a protein, or replicase )> RNA 3 (3a protein).

Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses.

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3'-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

The preparation of RNA-dependent RNA polymerase complex from virus infected plants.

Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into a tRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaic virus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solubilized membrane bound fractions. We accidentally found that these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity. Similar activity could also be found in other plants tested. Alternately, the membrane bound fraction could be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation (40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerol gradient fractionation was performed. Activity was tested through the incorporation of [alpha-32P]UTP using endogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractions contained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were found to have a double-stranded from through further analysis of the RNase protection assay.