Chemical cleavage of 5′-linked protein from tobacco ringspot virus genomic RNAs and characterization of the protein–RNA linkage

Abstract

Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5′-linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5′ nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting toin vivolabeling with32P,in vitroradioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5′ region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3′-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5′-oligo) by its sensitivity to proteinase. In a presumed β-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5′-oligo. Phosphatase-treated 5′-oligo accepted 5′-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5′ terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5′ uridylate of each genomic RNA.