Berkhout B, Klaver B, Das A T
Academic Medical Center, University of Amsterdam, Department of Human Retrovirology, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
Nucleic Acids Res. 1997 Mar 1;25(5):940-7. doi: 10.1093/nar/25.5.940.
The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.
HIV-1 RNA基因组的5'端和3'端形成一个重复（R）元件，该元件编码一种双茎环结构（TAR和多聚腺苷酸发夹结构）。对不同人类和猿猴免疫缺陷病毒中的多聚腺苷酸发夹结构进行系统发育分析表明，螺旋结构的热力学稳定性经过了微调。我们之前证明，具有稳定或不稳定发夹结构的突变HIV-1基因组在复制方面严重受损。在本研究中，我们发现具有不稳定多聚腺苷酸发夹结构的突变体存在条件性缺陷。虽然在正常温度（37摄氏度）下感染时其复制能力下降，但该突变体在较低温度（33摄氏度）下比野生型病毒更具适应性。这种对温度依赖性复制缺陷的观察强调了这种RNA结构的稳定性对其功能至关重要。为了进一步深入了解所研究元件的关键序列和结构特征，我们对回复病毒进行了广泛分析。将具有稳定或不稳定发夹结构的病毒突变体作为起点，通过多次独立筛选获得具有补偿性突变的回复病毒。这两种突变体都通过获得补偿性突变，沿着不同途径回复到具有野生型稳定性的发夹结构。我们鉴定出19种具有改善复制特性的不同回复HIV-1形式，首次揭示了总序列图谱中一些与病毒复制兼容的峰值。这些实验还突出了RNA结构构建的一些一般原则。
