Infectious DNA molecules, capable of transforming chicken embryo fibroblasts, can be synthesized by the Rous sarcoma virus-associated reverse transcriptase in vitro. The optimal enzymatic conditions employed for infectious DNA synthesis also facilitate maximum synthesis of genome length DNA. Analysis of the DNA product synthesized by detergent-disrupted Rous sarcoma virus under these conditions indicates that DNA complementary to viral RNA (minus-strand DNA) is genome length in size, whereas DNA complementary to genome length minusstrand DNA (plus-strand DNA) appears as subgenomic-length molecules ranging between 300 and 3,500 nucleotides in length. These features of the DNA product synthesized by the Rous sarcoma virus reverse transcriptase in vitro are similar to those identified in the cytoplasm of cells shortly after infection and lend credence to studies of the mechanism of reverse transcription in vitro and their significance to proviral DNA synthesis in vivo.

Electron microscopic analysis of the DNA product synthesized by detergent-disrupted preparations of Rous sarcoma virus in vitro revealed the presence of several interesting molecular forms including covalently closed circular DNA. The identification of such circular DNA indicates that virions of retroviruses contain all the components necessary to facilitate the complete synthesis of mature forms of viral DNA and therefore provide a useful system to delineate the molecular mechanisms involved in their synthesis.

No abstract

The minus-strand component of double-stranded DNA synthesized by the avian oncornavirus RNA-directed DNA polymerase in vitro represents as much of the nucleotide sequences of the viral genome as single-stranded DNA synthesized in the presence of actinomycin D. Since actinomycin D inhibits doublestranded DNA formation, we propose that the principal effect of the drug on RNA-directed DNA synthesis is the reduction in synthesis of plus-strand DNA, thereby allowing single-stranded minus-strand DNA to accumulate. This effect, rather than increasing the extent of genome transcription, is responsible for the more uniform nature of the DNA transcripts synthesized in the presence of actinomycin D.