Das A T, van Dam A P, Klaver B, Berkhout B
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands.
Virology. 1998 May 10;244(2):552-62. doi: 10.1006/viro.1998.9124.
The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple regulatory elements that fold into stable hairpin structures. Because extensive secondary structure can block the scanning of ribosomes, an alternative mechanism for HIV translation seems feasible. To study the mechanism of HIV-1 mRNA translation, a start codon was introduced in the leader region that will usurp scanning ribosomes. This upstream AUG mutation (uAUG) inhibited HIV gene expression, indicating that HIV-1 mRNA translation occurs via the regular scanning mechanism. Revertant viruses with increased replication capacity were obtained upon prolonged culturing of the mutant virus. To our surprise, the introduced start codon had not been inactivated in these phenotypic revertants. Instead, these revertants contain additional mutations in the envelope (Env) protein that stimulated HIV-1 replication. These second-site Env mutations did not specifically overcome the gene expression defect of the uAUG mutant, as the replication capacity of other HIV-1 mutants with an unrelated defect could also be improved. The uAUG construct appears to be a unique tool in forced HIV-1 adaptation studies because the deleterious uAUG mutation is stably maintained in the progeny, yielding phenotypic revertants with second-site mutations elsewhere in the viral genome.
人类免疫缺陷病毒（HIV）RNA基因组未翻译的前导区含有多个调控元件，这些元件可折叠成稳定的发夹结构。由于广泛的二级结构会阻碍核糖体的扫描，因此HIV翻译的另一种机制似乎是可行的。为了研究HIV-1 mRNA翻译的机制，在前导区引入了一个起始密码子，该密码子将夺取扫描核糖体。这种上游AUG突变（uAUG）抑制了HIV基因表达，表明HIV-1 mRNA翻译是通过常规扫描机制进行的。在对突变病毒进行长时间培养后，获得了具有增强复制能力的回复病毒。令我们惊讶的是，引入的起始密码子在这些表型回复株中并未失活。相反，这些回复株在包膜（Env）蛋白中含有额外的突变，这些突变刺激了HIV-1的复制。这些第二位点Env突变并未特异性地克服uAUG突变体的基因表达缺陷，因为其他具有无关缺陷的HIV-1突变体的复制能力也可以得到改善。uAUG构建体似乎是HIV-1强制适应性研究中的一种独特工具，因为有害的uAUG突变在子代中稳定维持，产生了在病毒基因组其他位置具有第二位点突变的表型回复株。
