Mikkelsen Jacob Giehm, Rasmussen Søren Vestergaard, Pedersen Finn Skou
Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark.
Nucleic Acids Res. 2004 Jan 9;32(1):102-14. doi: 10.1093/nar/gkh159. Print 2004.
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.
逆转录病毒颗粒含有二聚体RNA基因组，该基因组作为通过逆转录生成双链DNA的模板。DNA合成过程中RNA链之间的转移受模板之间的序列相似性和二聚体RNA的结构特征共同控制。一种被认为有助于分子间识别和二聚体形成的“亲吻发夹”结构，此前被发现是重组的一个优先位点。为了研究发夹环-环互补性是否是这种重组偏好的主要决定因素，我们设计了一种基于共包装两种野生型或环修饰的小鼠白血病病毒载体RNA的新型5'前导序列重组检测方法。我们发现，在两个载体之一中插入一个替代的回文环会破坏位点定向模板转换，而具有互补非野生型回文的载体之间恢复了位点特异性。通过配对包含相同非回文环基序且不太可能通过环-环“亲吻”相互作用的载体RNA，我们发现在“亲吻发夹”位点没有重组偏好。在设计通过非回文环基序的碱基配对相互作用的载体对中，尽管序列同一性降低，我们在一种情况下恢复了发夹定向模板转换，而另一对载体则未能支持发夹定向重组。然而，对所有研究的载体组合的体外RNA二聚化分析表明，环修饰的病毒RNA之间的有效二聚体形成与“亲吻发夹”处的体内cDNA转移之间存在良好的相关性。我们的研究结果表明，野生型或非野生型发夹“亲吻”环之间的互补性对于位点特异性5'前导序列重组是必不可少的，但并不充分，并进一步支持了这样一种假设，即特定的“亲吻”环-环相互作用由互补序列引导并维持在逆转录病毒成熟的二聚体RNA中。
