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Updated: Aug 9, 2026

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
Published on: December 4, 2007
Retroviral transduction of oncogenic sequences involves viral DNA instead of RNA
1University of California, Department of Molecular Biology, Berkeley 94720.
Abstract:
We have studied whether the origin of retroviral onc genes, by transduction of sequences from cellular proto-onc genes, involves DNA or RNA recombination. By using altered Harvey sarcoma proviruses as models for transduction intermediates, we have investigated the mechanism of regeneration of transforming virus from truncated proviruses with only a single 5' long terminal repeat (LTR) but with a complete 5'-LTR-ras transforming gene. The Harvey proviruses were specifically altered to discriminate between virus regeneration by RNA template switching during reverse transcription, as has been postulated, and virus regeneration by DNA recombination with either helper virus or among elements of the defective provirus alone. For this purpose U3 elements of the Harvey proviral LTR, which are essential for replication but not for transcription, were deleted in vitro. Only proviral constructions with an intact or a nearly intact single LTR regenerated infectious Harvey sarcoma virus. Since all constructions transformed cells and produced identical RNAs, our results exclude a model of virus regeneration by switching of RNA templates during reverse transcription. We conclude that regeneration of infectious Harvey viruses from truncated provirus involved illegitimate recombination of cellular or cotransfected DNAs flanking the 5'-LTR-ras gene of Harvey sarcoma virus. Based on this and evidence from the literature, we propose that retroviral transduction proceeds by way of rare illegitimate recombinations between proviral and cellular DNAs.
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