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United States Department of Agriculture

Agricultural Research Service

Title: Molecular Cloning and Nucleotide Sequencing of the 3'-Terminal Region of Ryegrass Mosaic Virus Rna and in Vitro Expression of the Coat Protein Gene

Authors
item Salm, S - RANGE & FOREST INS-S AFRI
item Rey, M - UNI OF WITWATERSRAND S AF
item French, Roy
item Rabenstein, F - FED CTR/PLT BRD GERMANY
item Schubert, J - FED CTR/PLT BRD GERMANY

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 23, 1995
Publication Date: N/A

Interpretive Summary: Plant viruses can often placed in groups based on several physical and biological characteristics. Such groupings are useful because findings based on the study of one member of a group can be usually extended to other members of the group. Ryegrass mosaic virus (RgMV) and wheat streak mosaic virus (WSMV), both mite-transmitted viruses of cereals, have been placed in the Rymovirus sub-group of the Potyviridae. We sought to determine the sequence of RgMV and compare it to WSMV. RgMV sequences were cloned and expressed in bacteria. Protein made by bacteria reacted with antibodies to the virus, confirming that we had cloned the coat protein gene. The RgMV cDNA sequence differed greatly from that of WSMV suggesting that the Rymoviruses, as currently defined, may not be a useful grouping for predictive purposes. Possible protease cleavage sites for the mature coat protein were deduced by alignment of the RgMV amino acid sequences with those of other Potyviruses.

Technical Abstract: Complementary DNA to the 3'-terminal nucleotides of the genomes of a South African strain and a Danish strain of RgMV (RgMV-SA and RgMV-Dan respectively), were cloned and sequenced. Comparison with other sequenced potyviruses indicated that clones of both contained the 3'-non coding region (3'-NCR), the coat protein (CP) gene and part of the nuclear inclusion (NIb) protein gene. The sequences consisted of a continuous open reading frame (ORF) which probably starts upstream of the cloned region as is the case for other potyviruses whose genomes are expressed as polyproteins. Possible Q/L or E/A protease cleavage sites for the N terminus of the CP were deduced by alignment of the amino acid sequences with those of other potyviruses. Cleavage at Q/L would yield proteins of 416 or 270 amino acids and calculated Mr of 47 or 30 kD. Cleavage at E/A would yield a protein of 342 amino acids and calculated Mr of 38 kD. The RgMV isolates showed cross reactivity to each other when tested with monoclonal antibodies raised against the Danish strain. RgMV-SA exhibited no cross reactivity with antisera against other Rymoviruses, but both RgMV strains did react with antisera to some Potyviruses. The RgMV-SA cDNA was cloned into an expression vector, pUEX, and a fusion protein of 69 kD was obtained which reacted strongly to anti-RgMV-SA antiserum. Alignment of the predicted amino acid sequences of RgMV-SA and RgMV-Dan with those of other Potyvirus and Rymovirus members showed limited identity, indicating that RgMV is a distinct virus.

Last Modified: 10/1/2014
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