McWilliams Mary Jane, Julias John G, Sarafianos Stefan G, Alvord W Gregory, Arnold Edward, Hughes Stephen H
HIV-Drug Resistance Program, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland 21702-1201, USA.
J Virol. 2003 Oct;77(20):11150-7. doi: 10.1128/jvi.77.20.11150-11157.2003.
The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5' end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5' end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5' end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.
逆转录病毒逆转录酶（RT）的核糖核酸酶H活性在病毒基因组RNA被复制成DNA后将其降解，去除用于起始负链DNA合成的tRNA，并生成和去除用于起始正链DNA合成的多聚嘌呤序列（PPT）引物。去除tRNA以及生成和去除PPT引物的切割必须具有特异性，以产生末端适合整合到宿主细胞基因组中的线性病毒DNA。1型人类免疫缺陷病毒（HIV-1）RT与源自PPT的RNA/DNA双链体形成复合物的晶体结构显示，PPT的5'端偏离传统的沃森-克里克碱基配对。这种不寻常的结构可能在HIV-1 RT对PPT的正确识别中起作用。我们在PPT的5'端进行了取代突变，并确定了它们对病毒滴度的影响。结果表明，在单轮试验中，PPT 5'端的单突变和双突变对病毒复制有适度影响。更复杂的突变对病毒滴度有更强的影响。对用突变病毒感染细胞产生的双长末端重复环连接的分析表明，这些突变影响核糖核酸酶H活性，导致病毒DNA上保留PPT序列。所测试的突变体优先保留PPT的特定片段，表明对切割特异性有影响。这些结果表明，PPT的结构特征对其在体内的识别和切割很重要。
