Characterization of the nucleic acid product of the visna virus RNA dependent DNA polymerase

The mechanism of nucleic acid synthesis and the nature of the nucleic acid products transcribed by the visna virus RNA dependent DNA polymerase have been investigated. Purified virus preparations activated maximally by .05% NP40 detergent catalyzed the linear incorporation of radioactive nucleoside precursor into acid insoluble product for 60 min and at a decreasing rate thereafter. Analysis of nucleic; acid products by S1 nuclease digestion, hydroxylapatite chromatography, and ultracentrifugation in cesium sulfate, indicate that the reaction proceeds in two steps. The initial step is transcription of a single-stranded DNA bound to RNA template. The second step involves synthesis of double-stranded DNA and this reaction can be blocked with actinomycin D. The DNA synthesized in the presence of actinomycin D contains sequences complementary to the entire viral genome. The double-stranded DNA product consists predominantly of a small molecular weight species with a sedimentation coefficient of 4S and an analytical complexity, measured by reassociation kinetics, of approximately 2-4 × 10^-3 mole-sec/liter. The visna polymerase therefore preferentially transcribes a limited portion of the viral genome into doublestranded DNA in vitro.