Polyadenylylation of reovirus proteins. Analysis of the RNA bound to structural proteins

The structure of the oligo(A) covalently bound to proteins in purified reovirions (Carter, C. A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3087-3091) has been characterized. The major adenylylated structural protein precipitated with immune serum is outer shell protein μ(1C), indicating that this polypeptide is the major adenylylated protein in virions. Polypeptides μ(1C) and a minor protein, viii, both contain two types of adenosine linkages. Oligo(A) is bound to viral proteins through the 3'-terminus of the RNA and ADP-ribose is bound through the ribose moiety. Eighteen per cent of the radioactivity in ¹²⁵I-labeled peptides of μ(1C) (derived by pronase digestion of proteins labeled by iodination of intact virions) bound to oligo(dT) cellulose in 0.5 M KCl. These peptides exhibited a net negative charge of -8 when analyzed by ion exchange chromatography. The adenylylated residues in polypeptide μ(C1) were shown to be susceptible to phosphorylation by polynucleotide kinase, indicating that protein is attached to the oligonucleotide at a position other than the 5'-end, possibly at the 3'-phosphate terminus of the RNA. This conclusion was supported by the results of experiments examining the susceptibility of RNA in the oligonucleotidyl protein to exonucleases with known group specificities: The oligo(A) associated with protein viii was resistant to digestion with snake venom exonuclease and susceptible to digestion with spleen phosphodiesterase. Alkaline and enzymatic hydrolysis also indicated that 15 to 25% of the [³H]adenosine radioactivity in both polypeptides was present as ADP-ribose of average chain length ≃ 1.3. The present experiments provide the first direct evidence that novel forms of adenylylated protein are present in the capsid of an animal virus.