Ribonucleotides in infectious bovine rhinotracheitis virus DNA

Infectious bovine rhinotracheitis (IBR) virus was grown in the presence of 5-3H-uridine in a continuous line of bovine kidney cells. 5-3H-uridine was found to be associated with viral nucleocapsids. Furthermore, purification of the viral nucleic acid present in nucleocapsids illustrated that 5-3H-uridine was part of the viral nucleic acid. Purification of viral DNA from infected cells also indicated that 5-3H-uridine was associated with viral nucleic acid possibly as ribonucleotides. The label was identified as RNA by measuring its susceptibility to RNase and analysis of the bases. Short pulses with 5-3H-uridine, resulted in labelled nucleic acid which was extremely sensitive to RNase and alkali but resistant to DNase. Nucleotide analysis indicated that after short pulses all the radioactivity was associated with the base uracil whereas upon longer labelling periods a large percentage of the label was associated with cytosine. However even if viral DNA was isolated from nucleocapsids there was still some radioactivity associated with uracil. Sedimentation of heat denatured 5-3H-uridine label viral nucleic acid in CS2SO4 indicated that the label sedimented at a density of single stranded DNA suggesting that the ribonucleotides are covalently linked to the viral DNA.