Initiation of DNA Synthesis by the Avian Retrovirus Reverse Transcriptase In Vitro: Nature and Location of the Oligodeoxycytidylic Acid Primer Binding Site

Abstract

We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC) 12-18 to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA) 12-18 nor oligo(dG) 12-18 was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC) 12-18 binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC) 12-18 binds to a site approximately 2,000 to 3,000 nucleotides from the 3′ terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3′ terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC) 12-18 appears to be in the vicinity of the 3′ end of the env gene and the 5′ end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.