Berkhout B, van Wamel J L
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands.
RNA. 2000 Feb;6(2):282-95. doi: 10.1017/s1355838200991684.
The untranslated leader of the RNA genome of the human immunodeficiency virus type 1 (HIV-1) encodes multiple signals that regulate distinct steps of the viral replication cycle. The RNA secondary structure of several replicative signals in the HIV-1 leader is critical for function. Well-known examples include the TAR hairpin that forms the binding site for the viral Tat trans-activator protein and the DIS hairpin that is important for dimerization and subsequent packaging of the viral RNA into virion particles. In this study, we present evidence for the formation of a tertiary structure by the complete HIV-1 leader RNA. This conformer was recognized as a fast-migrating band on nondenaturing polyacrylamide gels, and such a migration effect is generally attributed to differences in compactness. Both the 5' and 3' domains of the 335-nt HIV-1 leader RNA are required for the formation of the compact RNA structure, and the presence of several putative interaction domains was revealed by an extensive analysis of the denaturing effect of antisense DNA oligonucleotides. The buffer conditions and sequence requirements for conformer formation are strikingly different from that of the RNA-dimerization reaction. In particular, the conformer was destabilized in the presence of Mg2+ ions and by the viral nucleocapsid (NC) protein. The presence of a stable RNA structure in the HIV-1 leader was also apparent when this RNA was used as template for reverse transcription, which yielded massive stops ahead of the structured leader domain. Formation of the conformer is a reversible event, suggesting that the HIV-1 leader is a dynamic molecule. The putative biological function of this conformational polymorphism as molecular RNA switch in the HIV-1 replication cycle is discussed.
人类免疫缺陷病毒1型（HIV-1）RNA基因组的未翻译前导序列编码多种信号，这些信号调控病毒复制周期的不同步骤。HIV-1前导序列中几个复制信号的RNA二级结构对其功能至关重要。著名的例子包括形成病毒Tat反式激活蛋白结合位点的TAR发夹结构，以及对病毒RNA二聚化和随后包装到病毒粒子中很重要的DIS发夹结构。在本研究中，我们提供了完整的HIV-1前导RNA形成三级结构的证据。这种构象异构体在非变性聚丙烯酰胺凝胶上被识别为一条快速迁移的条带，这种迁移效应通常归因于紧密程度的差异。335个核苷酸的HIV-1前导RNA的5'和3'结构域对于紧密RNA结构的形成都是必需的，并且通过对反义DNA寡核苷酸变性效应的广泛分析揭示了几个假定的相互作用结构域的存在。构象异构体形成的缓冲液条件和序列要求与RNA二聚化反应的显著不同。特别是，在Mg2+离子存在下以及病毒核衣壳（NC）蛋白作用下，构象异构体会变得不稳定。当该RNA用作逆转录模板时，HIV-1前导序列中稳定RNA结构的存在也很明显，这在结构化前导结构域之前产生大量终止。构象异构体的形成是一个可逆事件，表明HIV-1前导序列是一个动态分子。本文讨论了这种构象多态性作为HIV-1复制周期中分子RNA开关的假定生物学功能。
