Complementarity-directed RNA dimer-linkage promotes retroviral recombination in vivo.
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.
Mikkelsen
Jacob Giehm
JG
Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark.
Rasmussen
Søren Vestergaard
SV
Pedersen
Finn Skou
FS
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D019281:Dimerization; D005822:Genetic Vectors / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D013698:Templates, Genetic; D014162:Transfection; D019065:Virus Assembly
D020121:5' Untranslated Regions; D012367:RNA, Viral