Joshi Pheroze, Prasad Vinayaka R
Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
J Virol. 2002 Jul;76(13):6545-57. doi: 10.1128/jvi.76.13.6545-6557.2002.
RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers (70.8,13, 70.15, 80.55,65, 70.28, 70.28t34, and 1.1) were selected based on binding constants (K(d)) and the degree of inhibition of RT in vitro (50% inhibitory concentration [IC(50)]). These six aptamers were each stably expressed in 293T cells followed by transfection of a molecular clone of HIV(R3B). Analysis of the virion particles revealed that the aptamers were encapsidated into the virions released and that the packaging of the viral genomic RNA or the cognate primer, tRNA(Lys)(3), was apparently unaffected. Infectivity of virions produced from 293T cell lines expressing the aptamers, as measured by infecting LuSIV reporter cells, was reduced by 90 to 99.5% compared to virions released from cells not expressing any aptamers. PCR analysis of newly made viral DNA upon infection with virions containing any of the three aptamers with the strongest binding affinities (70.8,13, 70.15, and 80.55,65) showed that all three were able to form the minus-strand strong-stop DNA. However, virions with the aptamers 70.8 and 70.15 were defective for first-strand transfer, suggesting an early block in viral reverse transcription. Jurkat T cells expressing each of the three aptamers, when infected with HIV(R3B), completely blocked the spread of HIV in culture. We found that the replication of nucleoside analog RT inhibitor-, nonnucleoside analog RT inhibitor-, and protease inhibitor-resistant viruses was strongly suppressed by the three aptamers. In addition, some of the HIV subtypes were severely inhibited (subtypes A, B, D, E, and F), while others were either moderately inhibited (subtypes C and O) or were naturally resistant to inhibition (chimeric A/D subtype). As virion-encapsidated TRTIs can predispose virions for inhibition immediately upon entry, they should prove to be efficacious agents in gene therapy approaches for AIDS.
通过指数富集配体系统进化（SELEX）获得的、对1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）具有特异性的RNA适配体，以高亲和力结合于模板-引物裂隙处并抑制其活性。为了确定此类模板类似物RT抑制剂（TRTIs）抑制HIV-1复制的潜力，10种适配体与侧翼自切割核酶一起表达，以产生侧翼序列最少的适配体RNA转录本。从中，根据结合常数（K(d)）和体外RT抑制程度（50%抑制浓度[IC(50)]）选择了6种适配体（70.8、13、70.15、80.55、65、70.28、70.28t34和1.1）。这6种适配体分别在293T细胞中稳定表达，随后转染HIV（R3B）分子克隆。对病毒粒子的分析表明，适配体被包裹在释放的病毒粒子中，病毒基因组RNA或同源引物tRNA(Lys)(3)的包装显然未受影响。通过感染LuSIV报告细胞来测量，与未表达任何适配体的细胞释放的病毒粒子相比，表达适配体的293T细胞系产生的病毒粒子的感染性降低了90%至99.5%。对感染含有三种结合亲和力最强的适配体（70.8、13、70.15和80.55、65）之一的病毒粒子后新合成的病毒DNA进行PCR分析表明，所有这三种适配体都能够形成负链强终止DNA。然而，含有适配体70.8和70.15的病毒粒子在第一链转移方面存在缺陷，这表明病毒逆转录早期受阻。表达这三种适配体中每一种的Jurkat T细胞在感染HIV（R3B）时，完全阻断了HIV在培养物中的传播。我们发现，这三种适配体强烈抑制核苷类似物RT抑制剂、非核苷类似物RT抑制剂和蛋白酶抑制剂耐药病毒的复制。此外，一些HIV亚型受到严重抑制（A、B、D、E和F亚型），而其他亚型则受到中度抑制（C和O亚型）或天然抗抑制（嵌合A/D亚型）。由于病毒粒子包裹的TRTIs可使病毒粒子在进入时立即易于受到抑制，它们应被证明是艾滋病基因治疗方法中的有效药物。
