In vitro Translation of the Bipartite Genomic RNA of Pea Early Browning Virus

Hughes, Gillian; Davies, Jeffrey W.; Wood, K.R.

doi:10.1099/0022-1317-67-10-2125

Cited by 16 publications

(12 citation statements)

References 26 publications

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“…140000 and 170000 (140K and 170K, Mayo et al, 1976;Fritsch et al, 1977) and that the larger protein may arise by the readthrough of a 'leaky' termination codon (Pelham, 1979). Similar studies using PEBV indicate that proteins of 134K and 165K are produced in the same manner (Hughes et al, 1986). A third protein of mol.…”

Section: Introductionsupporting

confidence: 54%

The Complete Nucleotide Sequence of Tobacco Rattle Virus RNA-1

Hamilton

Boccara

Robinson³

et al. 1987

Journal of General Virology

120

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SUMMARYThe complete nucleotide sequence of tobacco rattle virus (TRV) strain SYM RNA-1 was determined from a series of overlapping cDNA clones, cDNA prepared by primer extension was used to determine the exact 5' terminus. The RNA sequence was 6791 nucleotides in length and contained four open reading frames (ORFs). The ORF nearest the 5' terminus coded for a polypeptide of predicted tool. wt. 134000 (134K) and terminated at an opal (UGA) stop codon. Readthrough of this stop codon would result in the production of a protein of 194K. The gene for a 29K polypeptide started one base beyond the 194K stop codon and, in turn, was followed by the gene for a 16K protein at the 3' end of RNA-1. Amino acid comparisons of the 194K protein with the putative replicase of tobacco mosaic virus showed three regions of strong homology, suggesting that the 134K and 194K proteins were similarly involved in virus replication. The 5' terminal sequences of both genome RNA species of TRV strains ORY, N5 and PRN together with that of-SYM RNA-2 were also determined. Alignments of these sequences showed that there was a 22 base repeated sequence close to the 5' terminus in all these RNA species. It was also shown that the 5' terminus of RNA-1 was homologous with the same region in RNA-2.

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Section: Introductionsupporting

confidence: 54%

The Complete Nucleotide Sequence of Tobacco Rattle Virus RNA-1

Hamilton

Boccara

Robinson³

et al. 1987

Journal of General Virology

120

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“…As an additional control, total RNA from a virus-resistant plant (54. 3 The contiguous sequence is wild-type 54K, and the numbers are from the sequence of a full-length DNA copy of PEBV RNA1. Amino acid residues that differ from wild-type appear above the amino acid sequence line (in single-letter code).…”

Section: Resultsmentioning

confidence: 99%

“…PEBV RNA1 encodes two proteins with molecular masses of 141 and 201 kDa, which share amino acid sequence homology with the TMV 126-kDa and 183-kDa replicase proteins. These proteins are NH2-coterminal, the larger protein being expressed by the read-through of an opal (UGA) termination codon at the COOH terminus of the 141-kDa protein (1,3). Additionally, PEBV RNA1 encodes a 30-kDa protein with homology to the TMV 30-kDa cell-to-cell spread protein.…”

mentioning

confidence: 99%

Plants transformed with a region of the 201-kilodalton replicase gene from pea early browning virus RNA1 are resistant to virus infection.

MacFarlane

Davies²

1992

Proc. Natl. Acad. Sci. U.S.A.

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The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicoiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/mi, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the 16 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication.Pea early browning virus (PEBV) is a member of the Tobravirus group of plant viruses, which have a genome consisting of two positive-sense RNAs separately encapsidated in rodshaped particles. Analysis of the sequence of the viral RNAs revealed that the genetic organization of the larger RNA (RNA1) of PEBV resembles that of the monopartite virus tobacco mosaic virus (TMV) (1, 2). PEBV RNA1 encodes two proteins with molecular masses of 141 and 201 kDa, which share amino acid sequence homology with the TMV 126-kDa and 183-kDa replicase proteins. These proteins are NH2-coterminal, the larger protein being expressed by the read-through of an opal (UGA) termination codon at the COOH terminus of the 141-kDa protein (1,3). Additionally, PEBV RNA1 encodes a 30-kDa protein with homology to the TMV 30-kDa cell-to-cell spread protein. However, whereas the 3' proximal gene of TMV encodes the virus coat protein, this region in PEBV RNA1 encodes a cysteine-rich 12-kDa protein. PEBV coat protein is encoded by the 5' proximal gene of RNA2, which also encodes putative 29.6-kDa and 23-kDa proteins of unknown function (4).Closer inspection of the PEBV RNA1 sequence revealed that the COOH-terminal region of the 201-kDa protein could potentially be expressed separately as a 54-kDa protein by translation of an internal open reading frame (ORF) initiating at an AUG codon situated 147 nucleotides (encoding 49 amino acids) downstream of the termination codon (nucleotide 3866) for the 141-kDa protein. The PEBV ORF encoding the 54-kDa protein, designated 54K ORF,t encodes amino acid sequences that are highly conserved among the RNAdependent RNA polymerases from a wide range of plant, animal, and bacterial viruses. Several studies have presented detailed alignments of these proteins and identified a number of highly conserved sequence motifs that presumably ar...

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“…In vitro translation of the two genomic PEBV RNAs has shown that the coat protein is very inefficiently translated from RNA2 [4]. An explanation of this could be that the coat protein is translated from a subgenomic RNA derived from RNA2.…”

mentioning

confidence: 99%

“…Although in vitro translation of isolated PEBV RNA2 has not revealed synthesis of a protein of this size [4], some features indicate that ORF225 could encode a protein, which then should be produced from a subgenomic RNA in vivo. In TRV-TCM an ORF encoding a putative protein of 255 amino acids with a molecular mass of The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X15883-ORF 2t2, ORF 255.…”

mentioning

confidence: 99%