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United States Department of Agriculture

Agricultural Research Service

Title: Sequence Requirements for Translation Initiation of Rhopalosiphum Padi Virus Orf2

Authors
item Domier, Leslie
item McCoppin, Nancy
item Darcy, Cleora - UNIV OF ILLINOIS

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 5, 2000
Publication Date: N/A

Interpretive Summary: Barley yellow dwarf viruses (BYDVs) cause the most damaging virus disease of cereal crops. The viruses are spread from infected plants to healthy plants by aphids. Previous experiments have shown that insecticides can reduce the amount of damage caused by BYDVs in oat and wheat fields by limiting secondary spread of the viruses. The aphids that transmit BYDVs also can be infected with viruses. One of these insect viruses, Rhopalosiphum padi virus (RhPV), infects the aphid species that are the most common vectors of BYDVs in the central United States. RhPV infects a relatively narrow range of insects, which makes it suitable for use as a biological control agent that could replace chemical insecticides for controlling aphids in cereal crops. As a first step towards developing biological insecticides based on RhPV, we have determined how RhPV expresses the proteins that package and protect its small chromosome. The results of these experiments showed that RhPV employs a very unusual strategy to express its genes. This information will be useful to other scientists who are interested in developing virus-based insecticides and scientists who are studying basic mechanisms of gene expression. These experiments have allowed us to begin to design modified viral chromosomes that some day could be used as highly targeted biological insecticides.

Technical Abstract: Rhopalosiphum padi virus (RhPV) is an insect-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. The sequence for ORF2 lacks an obvious initiation codon, but an out-of- frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletions of sequences more than 300 nt upstream of the 5 border of ORF2 had no effect on translation initiation. Deletions extending from 220, 120 and 31 nt upstream of ORF2 abolished translation initiation. Changes in the out-of-frame AUG codon reduced, but did not inhibit translation initiation. Mutations that included the first and second codons of ORF2 failed to initiate protein synthesis. Deletions that started with the third codon and extended downstream showed reduced, but significant levels of translation. Deletions that started with the ninth codon translated normally. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to second codon of ORF2. These results show that the sequences necessary for translation initiation of the RhPV ORF2 include the first two codons and extend about 300 not upstream. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.

Last Modified: 10/20/2014
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