Published online 9 January 2004
Nucleic Acids Research, 2004, Vol. 32, No. 1 102-114
© 2004 Oxford University Press
Complementarity-directed RNA dimer-linkage promotes retroviral recombination in vivo
1 Department of Molecular Biology and 2 Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus, Denmark
*To whom correspondence should be addressed at Department of Molecular Biology, University of Aarhus, C. F. Moellers Allé, Building 130, DK-8000 Aarhus C, Denmark. Tel: +45 894 22614; Fax: +45 861 96500; Email: fsp{at}mb.au.dk
Present address:
Jacob Giehm Mikkelsen, Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
Retroviral particles contain a dimeric RNA genome, which serves as template for the generation of double-stranded DNA by reverse transcription. Transfer between RNA strands during DNA synthesis is governed by both sequence similarity between templates and structural features of the dimeric RNA. A kissing hairpin, believed to facilitate intermolecular recognition and dimer formation, was previously found to be a preferred site for recombination. To investigate if hairpin loop–loop-complementarity is the primary determinant for this recombination preference, we have devised a novel 5' leader recombination assay based upon co-packaging of two wild-type or loop-modified murine leukemia virus vector RNAs. We found that insertion of an alternative palindromic loop in one of the two vectors disrupted site-directed template switching, whereas site-specificity was restored between vectors with complementary non-wild-type palindromes. By pairing vector RNAs that contained identical non-palindromic loop motifs and that were unlikely to interact by loop–loop kissing, we found no preference for recombination at the kissing hairpin site. Of vector pairs designed to interact through base pairing of non-palindromic loop motifs, we could in one case restore hairpin-directed template switching, in spite of the reduced sequence identity, whereas another pair failed to support hairpin- directed recombination. However, analyses of in vitro RNA dimerization of all studied vector combinations showed a good correlation between efficient dimer formation between loop-modified viral RNAs and in vivo cDNA transfer at the kissing hairpin. Our findings demonstrate that complementarity between wild-type or non-wild-type hairpin kissing loops is essential but not sufficient for site-specific 5' leader recombination and lend further support to the hypothesis that a specific ‘kissing’ loop–loop interaction is guided by complementary sequences and maintained within the mature dimeric RNA of retroviruses.
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