Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/14/2001
Publication Date: N/A
Technical Abstract: Rhopalosiphum padi virus (RhPV) is a member of a newly defined family of insect-infecting viruses. These viruses possess positive-sense single-stranded RNA genomes of approximately 10 kb that contain two large open reading frames (ORFs) that express two polyproteins where the 5'- and 3'-proximal ORFs encode the nonstructural and capsid proteins, respectively. Both RhPV ORFs are preceded by over 500 nt of noncoding RNA that we have shown to function as internal ribosome entry sites (IRESs) in insect cells. The intergenic IRES (IG-IRES) is relatively short, 186 nt, and includes the first two codons of ORF2. Like most IRESs, the RhPV IG-IRES, facilitates the cap-independent translation of viral RNAs and allows ribosomes to initiate translation from within rather than from the terminus of the RNA. Unlike most eukaryotic mRNAs, translation of RhPV ORF2 is initiated at a CCU. The first two codons of RhPV ORF2 (CCU and GCA) are part of an inverted repeat that forms a pseudoknot at the 3' border of the IRES that is required for translation initiation in vivo. To test the interaction of the RhPV IG-IRES with eukaryotic initiation components, asp labeled in vitro transcripts containing the IG-IRES were cross-linked to proteins in rabbit reticulocyte lysate. The labeled IRES specifically bound a 68-kDa protein that was not bound by control templates or the 5' IRES. Labeled wild-type IG-IRES was recruited into 48S preinitiation complexes, but truncated elements failed to enter the complexes. In vivo studies using a baculovirus-T7 RNA polymerase system confirmed that a loss of ability to enter the 48S complexes was associated with a loss of biological activity.