Electron microscopy of double-stranded RNA induced by turnip yellow mosaic virus and tobacco mosaic virus

Summary Double-stranded RNA isolated by phenol extraction from turnip yellow mosaic virus-infected chinese cabbage leaves and from tobacco mosaic virus-9nfected tobacco leaves was rotary shadowed and examined in the electron microscope. The TYMV and TMV molecules are similar in appearance, having uniform width and a linear configuration similar to that previously described for double-stranded RNA and double-stranded DNA molecules. More than 99.5% of the molecules of each virus fall within the range 0.1 to 2.2 , there being a predominance of smaller molecules in both cases (TYMV mean=0.24 , TMV mean 0.42 ). The mode of the larger molecules of TYMV 1.92 and of TMV 1.8 . These values are close to the expected lengths of whole molecules, calculated from biophysical data. Apparently branched molecules were observed in preparations of both TYMV and TMV double-stranded RNA. It was found, however, that the number of such branches per unit length of RNA decreases with a decrease in density of the RNA in the fields examined.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex

The approximately 150 nt tRNA-like structure present at the 3' end of each of the brome mosaic virus (BMV) genomic RNAs is sufficient to direct minus-strand RNA synthesis. RNAs containing mutations in the tRNA-like structure that decrease minus-strand synthesis were tested for their ability to interact with RdRp (RNA-dependent RNA polymerase) using a template competition assay. Mutations that are predicted to disrupt the pseudoknot and stem B1 do not affect the ability of the tRNA-like structure to interact with RdRp. Similarly, the +1 and +2 nucleotides are not required for stable template-RdRp interaction. Mutations in the bulge and hairpin loops of stem C decreased the ability of the tRNA-like structure to interact with RdRp. Furthermore, in the absence of the rest of the BMV tRNA, stem C is able to interact with RdRp. The addition of an accessible initiation sequence containing ACCA3' to stem C created an RNA capable of directing RNA synthesis. Synthesis from this minimal minus-strand template is dependent on sequences in the hairpin and bulged loops.     

In vitro synthesis of turnip yellow mosaic virus coat protein in a wheat germ cell-free system

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.     

Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.     

Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu

Turnip yellow mosaic virus (TYMV) Val-RNA forms a complex with the peptide elongation factor Tu (EF-Tu) in the presence of GTP: the Val-RNA is protected by EF-Tu·GTP from non-enzymatic deacylation and nuclease digestion. The determination of the length of the shortest TYMV Val-RNA fragment that binds EF-Tu·GTP leads us to conclude that the valylated aminoacyl RNA domain equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T arm is sufficient for complex formation. Since the aminoacyl RNA domain is also sufficient for adenylation by the ATP(CTP):tRNA nucleotidyltransferase, an analogy can be drawn between these two tRNA-specific proteins.     

Inhibition of turnip yellow mosaic virus synthesis by pyrimidine analogues

The pyrimidine analogues 2-thiouracil, 2-thiouridine, 6-azauracial and 6-azauridine all inhibited the synthesis of turnip yellow mosaic virus (TYMV) and increased the synthesis of empty virus protein shells in infected Chinese cabbage leaf discs. Uracil and uridine reversed these effects. 2-Thiouracil also reduced the UTP pool in TYMV infected leaf discs. The results are consistent with the suggestion that these analogues or their in vivo derivatives affect virus synthesis by inhibiting the biosynthesis of uridylic acid, possibly by inhibiting orotidylic acid decarboxylase.     

Proteolytic origin of the 150-kilodalton protein encoded by turnip yellow mosaic virus genomic RNA.

Turnip yellow mosaic virus genomic RNA codes in vitro for two overlapping proteins, 150-kilodalton (150K protein) and 206-kilodalton (206K protein) proteins. The proteolytic maturation known to affect the 206K protein has been further characterized by in vitro translation assays in a reticulocyte lysate or wheat germ extract. Cleavage is inhibited at 37 degrees C and restored when the temperature is shifted to 30 or 25 degrees C. Temperature shift experiments are used here to demonstrate that the 150K protein and the previously characterized 78K protein are the two fragments resulting from a primary cleavage phenomenon that affects the 206K protein in a cotranslational manner under usual translation conditions. This processing is prevented by several cysteine and serine proteinase inhibitors.     

Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Summary Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4–5.8 with citrate buffer; concentration of poly-L-ornithine, 0.8–1.0 g/ml; concentration of TYMV, 0.2–1.0 g/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced 1 to 2x10⁶ TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.     

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.