Verhoef K, Berkhout B
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
J Virol. 1999 Apr;73(4):2781-9. doi: 10.1128/JVI.73.4.2781-2789.1999.
We previously constructed a large set of mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat with conservative amino acid substitutions in the activation domain. These Tat variants were analyzed in the context of the infectious virus, and several mutants were found to be defective for replication. In an attempt to obtain second-site suppressor mutations that could provide information on the Tat protein structure, some of the replication-impaired viruses were used as a parent for the isolation of revertant viruses with improved replication capacity. Sequence analysis of revertant viruses frequently revealed changes within the tat gene, most often first-site reversions either to the wild-type amino acid or to related amino acids that restore, at least partially, the Tat function and virus replication. Of 30 revertant cultures, we identified only one second-site suppressor mutation. The inactive Y26A mutant yielded the second-site suppressor mutation Y47N that partially restored trans-activation activity and virus replication. Surprisingly, when the suppressor mutation was introduced in the wild-type Tat background, it also improved the trans-activation function of this protein about twofold. We conclude that the gain of function measured for the Y47N change is not specific for the Y26A mutant, arguing against a direct interaction of Tat amino acids 26 and 47 in the three-dimensional fold of this protein. Other revertant viruses did not contain any additional Tat changes, and some viruses revealed putative second-site Tat mutations that did not significantly improve Tat function and virus replication. We reason that these mutations were introduced by chance through founder effects or by linkage to suppressor mutations elsewhere in the virus genome. In conclusion, the forced evolution of mutant HIV-1 genomes, which is an efficient approach for the analysis of RNA regulatory motifs, seems less suited for the analysis of the structure of this small transcription factor, although protein variants with interesting properties can be generated.
我们之前构建了一大组人类免疫缺陷病毒1型（HIV-1）调节蛋白Tat的突变体，这些突变体在激活域中具有保守的氨基酸替换。在感染性病毒的背景下对这些Tat变体进行了分析，发现有几个突变体在复制方面存在缺陷。为了获得能够提供Tat蛋白结构信息的第二位点抑制突变，一些复制受损的病毒被用作亲本，用于分离具有改善复制能力的回复病毒。回复病毒的序列分析经常揭示tat基因内的变化，最常见的是第一位点回复为野生型氨基酸或相关氨基酸，这至少部分恢复了Tat功能和病毒复制。在30个回复培养物中，我们仅鉴定出一个第二位点抑制突变。无活性的Y26A突变体产生了第二位点抑制突变Y47N，该突变部分恢复了反式激活活性和病毒复制。令人惊讶的是，当在野生型Tat背景中引入抑制突变时，它也使该蛋白的反式激活功能提高了约两倍。我们得出结论，Y47N变化所测得的功能增益并非Y26A突变体所特有的，这表明在该蛋白的三维折叠中，Tat的第26位和第47位氨基酸不存在直接相互作用。其他回复病毒未包含任何额外的Tat变化，一些病毒显示出推测的第二位点Tat突变，但这些突变并未显著改善Tat功能和病毒复制。我们推断这些突变是通过奠基者效应偶然引入的，或者是与病毒基因组其他位置的抑制突变连锁引入的。总之，突变HIV-1基因组的强制进化是分析RNA调节基序的有效方法，但似乎不太适合分析这种小转录因子的结构，尽管可以产生具有有趣特性的蛋白质变体。
