Yellow Mosaic Virus Genomic RNA Research Articles

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.

Aminoacylation identity switch of turnip yellow mosaic virus RNA from valine to methionine results in an infectious virus.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

The Complete Nucleotide Sequence of Bean Yellow Mosaic Virus Genomic RNA.

The complete nucleotide sequence of the RNA genome of bean yellow mosaic potyvirus (BYMV) has been determined. The RNA genome of BYMV is 9532 nucleotides long excluding the poly (A) tract. The RNA contains one open reading frame (ORF) of 9171 nucleotides encoding a large polypeptide of 3056 amino acids with a calculated Mr of 347, 571. Nine putative proteolytic cleavage sites, one by P1 protease, one by HC protease and seven by NIa protease, are identified by comparison with those identified for other sequenced potyviruses. The genetic organization of BYMV genome is proposed to be 5'UTR (190nt)-P1 (33K)/HC-Pro (52K)/P3 (41K)/6K1/CI (71K)/6K2/NIa-VPg (22K)/NIa-Pro (27K)/NIb (59K)/CP (31K)-3'UTR (171nt)-poly (A). Comparing the amino acid sequences of individual BYMV proteins with the corresponding proteins of other potyviruses, the BYMV proteins, excepting P1, have a high or moderate homology.

Proteolytic maturation of the 206-kDa nonstructural protein encoded by turnip yellow mosaic virus RNA

The longest open reading frame of turnip yellow mosaic virus genomic RNA (ORF-206) encodes a 206-kDa nonstructural protein. The most prominent in vitro translation products of ORF-206 are the full-length p206 and a shorter N-coterminal 150-kDa protein. We have confirmed these assignments by immunoprecipitation of in vitro translation products with antisera raised to N-terminal and C-terminal regions encoded by ORF-206. The mechanism by which the 150-kDa protein arises from ORF-206 was investigated by in vitro translation of deletion and substitution derivatives transcribed from pTYMC, a cDNA clone of TYMV RNA. The following observations demonstrate that the 150-kDa protein and a C-terminal 70-kDa protein arise from ORF-206 by autoproteolysis: (1) Two regions encoded by ORF-206 were necessary for the formation of the 150-kDa protein: a domain between amino acids 555 and 1051, postulated to encode a protease, and the region between amino acids 1253 and 1261, thought to constitute the protease recognition and/or cleavage site. (2) Mutants with substitutions between amino acids 1253 and 1261 that produce low levels of the 150-kDa protein in in vitro translations also have high levels of p206 and low levels of the 70-kDa protein. (3) The rate of formation of the 150-kDa protein is dilution insensitive, suggesting that proteolysis occurs mainly in cis.

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.

Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

The complete nucleotide sequence of turnip yellow mosaic virus (TYMV) genomic RNA has been determined on a set of overlapping cDNA clones using a sequential sequencing strategy. The RNA is 6318 nucleotides long, excluding the cap structure. The genome organization deduced from the sequence confirms previous results of in vitro translation. A novel open reading frame (ORF) putatively encoding a Pro-rich and very basic 69K (K = kilodalton) protein is detected at the 5' end of the genome. It is initiated at the first AUG codon on the RNA and overlaps the major ORF that encodes the non structural 206K (previously referred to as 195K) protein of TYMV; its function is unknown. Several amino acid consensus sequences already described among plant and animal viruses are also found in the TYMV-encoded polypeptides. A comparison with other viruses whose RNA sequence is known leads to the conclusion that TYMV belongs to the "Sindbis-like" supergroup of viruses and could be related to Semliki forest virus.

Analysis of the in vitro coding properties of the 3′ region of turnip yellow mosaic virus genomic RNA

The genomic RNA of turnip yellow mosaic virus (TYMV) codes in vitro for two proteins of 195,000 (195K) and 150,000 (150K) daltons initiated at the same site of the RNA. In the presence of yeast amber suppressor tRNA, synthesis of the 195K protein is reduced in favor of a 210,000-dalton (210K) protein. Using TYMV RNA sequence data and comparison of tryptic peptides of the 150K, 195K, 210K, and coat proteins, we postulate that the silent region between the 195K gene and the coat protein gene located in the 3′ region of TYMV genomic RNA is only 14 nucleotides long.

Post-Translational Proteolytic Cleavage of In Vitro-Synthesized Turnip Yellow Mosaic Virus RNA-Coded High-Molecular-Weight Proteins

In a reticulocyte lysate, turnip yellow mosaic virus genomic RNA directs the synthesis of two proteins with molecular weights of 150,000 (150K) and 195K. We present evidence that the larger protein is processed in vitro, after its completion, in at least three fragments. The NH(2)-terminal fragment (82K) and the COOH-terminal fragment (78K) have been well characterized by different methods. The fact that the 150K protein is not cleaved in vitro, although it contains the regions that are processed in the 195K protein, could be of fundamental biological significance for the expression of the viral genes: a single polypeptide chain could be processed in several ways, leading to different peptides with distinct biological activities.