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J Virol, March 1998, p. 2519-2525, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

Jacob Giehm Mikkelsen,1 Anders H. Lund,1 Karen Dybkær,1 Mogens Duch,1 and Finn Skou Pedersen1,2,*

Department of Molecular and Structural Biology1 and Department of Medical Microbiology and Immunology,2 University of Aarhus, DK-8000 Aarhus, Denmark

Received 14 August 1997/Accepted 13 November 1997

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439-1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

* Corresponding author. Mailing address: Department of Molecular and Structural Biology, University of Aarhus, C. F. Moellers Allé, Bldg. 130, DK-8000 Aarhus, Denmark. Phone: 45 89423188. Fax: 45 86196500. E-mail: fsp{at}mbio.aau.dk.




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