Cellular Protein Binds to Sequences near the 5′ Terminus of Potato virus X RNA That Are Important for Virus Replication

AbstractThe sequences in the 5′-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5′-NTR. These analyses showed that the 5′ region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1–46 at the 5′ terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10–13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5′ terminus of the viral genomic RNA