CNRS UMR 8015 Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, 4 Avenue de l'Observatoire, 75270 Paris cedex 06, France.
Biochimie. 2012 Jul;94(7):1481-9. doi: 10.1016/j.biochi.2012.02.009. Epub 2012 Feb 16.
The 5'UnTranslated Region (5'UTR) of HIV-1 genomic RNA, which precedes the Gag coding sequence, fulfills several roles during the lentivirus life cycle. This 335 nucleotides leader contains many stable structures that are crucial for the regulation of genetic expression at the level of transcription, splicing, and translation. In the late steps of the virus cycle, i.e. virions formation, the genomic RNA serves as propagated genome and its encapsidation in new particles relies on its ability to form non-covalent dimers. Dimerization is proposed to be initiated by the intermolecular pairing of a self-complementary sequence located in the apical loop of the DIS hairpin (Dimer Initiation Sequence). The regulation of this phenomenon and the extraordinary stability of the dimers imply that structural elements other than this kissing complex remain to be identified. Here, we show that swapping the Gag open reading frame (ORF) by reporter genes interferes with dimers formation efficiency. Importantly, the nature of the ORF alters specific structures of the 5'UTR. By using a systematic "SHAPE" approach, we pointed out that sequences within the Major Splice Site are involved in the dimerization process. Furthermore, by the use of an antisense oligonucleotide specific for the MSD associated to a SHAPE analysis of the 5'UTR structure, we demonstrated that interfering with the MSD results both in an impaired dimerization and in modifications of the 5'UTR structure. All together these data support a recently proposed model in which intramolecular base pairings are important determinants for the dimerization process. We further conclude that much care should be taken when comparing translation activity of reporter constructs with the viral situation.
HIV-1 基因组 RNA 的 5'非翻译区（5'UTR）位于 gag 编码序列之前，在慢病毒生命周期中发挥多种作用。这个 335 个核苷酸的先导含有许多稳定的结构，对于转录、剪接和翻译水平的遗传表达调控至关重要。在病毒周期的后期，即病毒粒子形成，基因组 RNA 作为增殖基因组，其在新颗粒中的包裹依赖于其形成非共价二聚体的能力。二聚化被提议由位于 DIS 发夹（二聚化起始序列）顶端环中的自我互补序列的分子间配对引发。这种现象的调节和二聚体的非凡稳定性意味着除了这个亲吻复合物之外，还有其他结构元件有待确定。在这里，我们表明通过报告基因交换 gag 开放阅读框（ORF）会干扰二聚体的形成效率。重要的是，ORF 的性质改变了 5'UTR 的特定结构。通过使用系统的“SHAPE”方法，我们指出主要剪接位点内的序列参与二聚化过程。此外，通过使用针对与 5'UTR 结构的 SHAPE 分析相关的 MSD 的反义寡核苷酸，我们证明干扰 MSD 会导致二聚化受损和 5'UTR 结构发生变化。所有这些数据都支持最近提出的模型，即分子内碱基配对是二聚化过程的重要决定因素。我们进一步得出结论，在比较报告基因构建体的翻译活性与病毒情况时，应格外小心。
