Powell M D, Levin J G
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
J Virol. 1996 Aug;70(8):5288-96. doi: 10.1128/JVI.70.8.5288-5296.1996.
At the 3' end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3' end of the PPT. In addition, we find that a hybrid consisting of a 15- or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5' end of the PPT have no effect on primer function, whereas the identity of bases at the 3' end is crucial. A primer containing only the 6 G residues from the 3' end of the wild-type PPT sequence and 9 bases of random sequence at the 5' end functions like a wild-type PPT. A short hybrid having a similar helical structure but a primary sequence different from that of the PPT is cleaved imprecisely, resulting in initiation of synthesis at multiple sites; however, total primer extension is close to the wild-type level. We conclude that helical structure as well as the presence of particular bases at the 3' end of the PPT is essential for PPT function.
在所有逆转录病毒基因组的3'端，都有一段短的、高度保守的序列，称为多聚嘌呤序列（PPT），它作为正链DNA合成的引物。我们已经确定了人类免疫缺陷病毒1型（HIV-1）PPT在体外引发的决定因素。我们发现，当PPT被去除并置于不同的核苷酸环境中时，新的引发位点会在PPT精确的3'端产生。此外，我们发现由一个15或20个核苷酸的RNA引物与一个35个核苷酸的DNA模板退火形成的杂交体，能够与HIV-1逆转录酶一起启动正链合成。因此，引发活性似乎不需要顺式作用元件。PPT 5'端的变化对引物功能没有影响，而3'端碱基的身份至关重要。一个仅包含野生型PPT序列3'端的6个G残基和5'端9个随机序列碱基的引物，其功能类似于野生型PPT。一个具有相似螺旋结构但一级序列与PPT不同的短杂交体被不精确切割，导致在多个位点启动合成；然而，总的引物延伸接近野生型水平。我们得出结论，螺旋结构以及PPT 3'端特定碱基的存在对PPT功能至关重要。
