Fuentes G M, Rodríguez-Rodríguez L, Fay P J, Bambara R A
Department of Microbiology & Immunology, University of Rochester, School of Medicine and Dentistry, New York 14642, USA.
J Biol Chem. 1995 Nov 24;270(47):28169-76. doi: 10.1074/jbc.270.47.28169.
A primary site for initiation of plus strand DNA synthesis in human immunodeficiency virus (HIV) corresponds to a 19-nucleotide-long purine rich sequence located just upstream of the U3 region, designated the polypurine tract (PPT). The HIV reverse transcriptase (RT) uses its RNase H activity to cut the genomic RNA after minus strand DNA synthesis. A plus strand PPT primer is formed, extended, and then removed. In vitro, the HIV-RT recognizes this primer specifically, using it much more efficiently than other RNA primers. However, the PPT still primes significantly less efficiently than DNA primers. The 19-nucleotide PPT primer is partially resistant to degradation when compared with other oligoribonucleotides. Prior to initiation of DNA synthesis, several nucleotides are removed by the RT from the 3' ends of some of the PPT primers. Cleavage is enhanced in the absence of dNTPs. We suggest that DNA synthesis suppresses primer degradation, so that primer extension and cleavage occur in proper sequence. As a result of 3' end degradation, PPT elongation products contain 5'-RNA segments from 16 to 19 nucleotides in length. These shorter segments are also generated from a longer transcript containing the PPT sequence, indicating that they are not created as a result of binding of the RT to the 5' end of the PPT oligoribonucleotide. Full-length and shorter versions of the PPT primers are cleaved from the extended DNA by RT. These experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT but that the ends of the primer are not well defined.
人类免疫缺陷病毒（HIV）中正向链DNA合成起始的主要位点对应于位于U3区域上游的一段19个核苷酸长的富含嘌呤的序列，称为多嘌呤序列（PPT）。HIV逆转录酶（RT）利用其核糖核酸酶H活性在负向链DNA合成后切割基因组RNA。形成、延伸并随后去除正向链PPT引物。在体外，HIV-RT能特异性识别该引物，使用它的效率比其他RNA引物高得多。然而，PPT引发的效率仍明显低于DNA引物。与其他寡核糖核苷酸相比，19个核苷酸的PPT引物对降解具有部分抗性。在DNA合成起始之前，RT从一些PPT引物的3'末端去除几个核苷酸。在没有脱氧核苷酸三磷酸（dNTPs）的情况下，切割作用增强。我们认为DNA合成会抑制引物降解，从而使引物延伸和切割按正确顺序发生。由于3'末端降解，PPT延伸产物包含长度为16至19个核苷酸的5'-RNA片段。这些较短的片段也来自包含PPT序列的较长转录本，这表明它们不是RT与PPT寡核糖核苷酸5'末端结合的结果。RT从延伸的DNA上切割下全长和较短版本的PPT引物。这些实验表明，HIV-RT在PPT区域生成引物具有特异性，但引物的末端并不明确。
