Structural Features of Encephalomyocarditis Virus RNA from Analysis of Reverse Transcription Products

Abstract

The presence in encephalomyocarditis (EMC) virus RNA of homonucleotide tracts 10 nucleotides or more in length has been investigated by testing the ability of homo‐oligodeoxynucleotides to prime DNA synthesis in the presence of reverse transcriptase from avian myeloblastosis virus. Neither (dC)₁₀ nor (dA)₁₀ promoted incorporation of [³H]deoxynucleotides into acid‐insoluble material but (dG)₁₀ and (dT)_12–18 were effective primers and produced DNA products approximately 2000 nucleotides in length. We conclude that there are single‐stranded oligo(rC) and oligo(rA) tracts in native EMC virus RNA at 37°C. Kinetic analysis indicated that oligo(dT) priming is similar to priming on ovalbumin mRNA and that it gives rise to only one DNA product per template molecule. Oligo(dG) priming appears to be complicated by self‐aggregation of the primer. Oligo(dT)‐primed and oligo(dG)‐primed DNA have both been separated on alkaline‐sucrose gradients into two peaks of which only the ‘heavier’ will hybridise to EMC virus RNA. Competitive hybridisation experiments indicate that the ‘heavy’ oligo(dT)‐primed and oligo(dG)‐primed DNA fractions hybridise to overlapping sequences of EMC virus RNA and place the priming regions of EMC virus RNA approximately 500 nucleotides apart during reverse transcription.