Kinetics of synthesis, structure and purification of avian sarcoma virus-specific DNA made in the cytoplasm of acutely infected cells

Abstract

We have characterized virus-specific DNA synthesized in the cytoplasm of quail tumor cells acutely infected with avian sarcoma virus, using rate-zonal sedimentation and polyacrylamide gel electrophoresis, in conjunction with molecular hybridization reagents specific for various regions of the viral genome. We have detected (−) strand DNA (complementary to the viral genome) within 45 minutes after infection, and we have observed the elongation of this strand, from 1 to 2 kb † † Abbreviations used: kb, kilobase; (−) strand, strand complementary to the viral genome; (+) strand, strand with chemical polarity identical to that of the viral genome; ASV, avian sarcoma virus; B77, Bratislava-77 strain of ASV; HH, LL etc., heavy-heavy and light-light DNA. to 8 to 10 kb in length, at the relatively slow rate of approximately 30 nucleotides/minute. (+) Strands of viral DNA (the same polarity as the viral genome) were also produced within 45 minutes after infection, indicating that completion of the (−) strand is not a prerequisite for synthesis of (+) strands. During the first 2 to 3 hours after infection, the principal species of (+) strand is a fragment of approximately 250 to 300 nucleotides, containing sequences specific for the 5′ and the 3′ termini of the viral genome. The linkage of sequences from the ends of the RNA genome in this short (+) strand was confirmed by the demonstration of a recognition site for endo R EcoRI near the middle of the strand. (+) Strands representing other regions of the genome (including the genes gag, pol, env and src) appear in gradually increasing quantities between 1.5 and 10 hours after infection; these strands are heterogeneous in size and generally longer (approx. 500 to 1500 nucleotides) than the 250 to 300 nucleotide (+) strand containing sequences from the termini of the viral genome. Homopolymeric tracts were not detected in either the (+) or (−) strands using affinity chromatography on oligo(dT)-cellulose columns; thus, the polyadenylic acid residues at the 3′ terminus of the viral genome are apparently added posttranscriptionally and not copied during RNA-directed DNA synthesis. We have exploited the structural features of linear viral DNA described here to purify subunit-length (−) strands from infected cells. The viral DNA was labeled by density with bromodeoxyuridine and freed of cellular DNA by cell fractionation, equilibrium centrifugation in cesium chloride gradients, and rate-zonal centrifugation in neutral sucrose gradients before and after denaturation. The purity and usefulness of the (−) strands were documented by the synthesis of 32P-labeled (+) strands of virus-specific DNA with Escherichia coli DNA polymerase I, using extremely small quantities of (−) strands as template (approx. 10 ng) and oligomers of calf thymus DNA as primers. The 32P-labeled DNA was shown to anneal specifically to viral DNA and to yield discrete fragments upon digestion with endo R EcoRI.