CHEMICAL CLEAVAGE OF 5'-LINKED PROTEIN FROM TOBACCO RINGSPOT VIRUS GENOMIC RNAS AND CHARACTERIZATION OF THE PROTEIN-RNA LINKAGE

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' nu cleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting to in vivo label ing with P-32, in vitro radioiodination, or separation of the two geno mic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyr ibonucleotide specific for the 5' region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3'-end-labeled and were fractio nated 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 p-eliminati on reaction that apparently was more rapid than phosphodiester cleavag e, incubation in alkaline sodium bicarbonate released a rapidly migrat ing product, 5'-oligo. Phosphatase-treated 5'-oligo accepted 5'-label in a polynucleotide kinase-catalyzed reaction, and uridylate was ident ified as the 5' terminal residue for both RNA 1 and RNA 2. Results fro m Edman degradation of the VPg suggest that the VPS is linked at serin e 5 to the 5' uridylate of each genomic RNA. (C) 1996 Academic Press, Inc.