DNA RECOMBINATION IS SUFFICIENT FOR RETROVIRAL TRANSDUCTION

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomolog ous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied i n terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA rec ombination and a 3' RNA recombination occurring during reverse transcr iption of viral and protooncogene RNA. Here we use two viral DNA const ructs to test the prediction of the DNA model that the 3' DNA recombin ation is achieved by conventional integration of a retroviral DNA 3' o f the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purpo rtedly essential polypurine tract (ppt) that precedes the 3' long term inal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally Linked at the 5' end to a retroviral LTR and artificially linked at the 3' end e ither directly (construct NdN) or by a cellular sequence (construct SU ) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end, Both constructs p ro, ed to be infectious, producing viruses at titers of 10(5) focus-fo rming units per ml. Sequence analysis proved that both viruses were co linear with input DNAs and that NdN virus lacked a ppt and the 5' 30 n ucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor th e complete LTR is essential for retrovirus replication. DNA recombinat ion explains the following observations by others that cannot be recon ciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental trans duction may invert sequences with respect to others, as expected for D NA recombination during transfection.