Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 [alpha]. Search for a function

The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase.Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha.. GTP against digestion by RNase A. By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP. The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop. In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A. It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha. This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.     

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.     

Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.     

Nucleotide sequence of turnip yellow mosaic virus coat protein mRNA

The primary structure of the coat protein messenger RNA of turnip yellow mosaic virus is presented. This sequence is the first complete nucleotide sequence of the coat protein messenger of a plant virus to be reported. The coding region, consisting of 567 nucleotides, is flanked by a 5′ noncoding region of 19 nucleotides (not including the initiation codon and the cap structure) and by a 3′ noncoding region of 109 nucleotides (including the termination signal). The coat protein mRNA has a base composition identical to that of the genome RNA with, in particular, the same high content in cytosine (38%). The codons that govern the incorporation of amino acids into the coat protein are nonrandomly utilized: >50% of the time the third base of the codons used is a cytosine. This pattern of codon preference is particularly marked for Leu, lie, Val, Thr and Cys.     

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.     

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.     

Seed transmission of turnip yellow mosaic virus in winter turnip and winter oilseed rapes

This is the first record of seed transmission of turnip yellow mosaic virus (TYMV) in oilseed and turnip rapes. The seed transmission of TYMV in a naturally infected winter turnip rape (Brassica napus var. silvestris) cultivar Perko PVH was investigated. By ELISA 1.6%, 3.2% and 8.3% seed transmission of the virus was found in seed of plants from three localities. The proportion of infected seeds produced by artificially infected plants of winter oilseed rape (Brassica napus ssp. oleifera) and winter turnip rape cultivars was determined. The virus transmission rate, expressed as the proportion of virus-infected plants which germinated from the seed was for the oilseed rape cvs Jet Neuf 0.1%, Solida 0.4%, Silesia 0.8%, Darmor 1.2%, SL-507 0.2%, SL-509 0.0% and for the winter turnip rape cv. Perko 1.5%. ELISA cannot be used in direct tests on bulk seed lots to estimate proportion of infected seed, but must be used on germinated seedlings.