Robust Suppression of HIV Replication by Intracellularly Expressed Reverse Transcriptase Aptamers Is Independent of Ribozyme Processing
Robust Suppression of HIV Replication by Intracellularly Expressed Reverse Transcriptase Aptamers Is Independent of Ribozyme Processing
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DOI：
10.1038/mt.2012.158
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发表时间：
2012-12-01
影响因子：
12.4
通讯作者：
Burke, Donald H.
中科院分区：
文献类型：
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作者：
Lange, Margaret J.;Sharma, Tarun K.;Burke, Donald H.
结合人类免疫缺陷病毒1（HIV-1）逆转录酶（RT）的RNA适体也抑制病毒复制，使它们作为用于剖析病毒发病机制的治疗候选物和潜在工具而具有吸引力。然而，还没有很好地理解适体表达背景和细胞RNA途径如何控制适体积累和净抗病毒生物活性。使用先前描述的表达盒，其中适体两侧是两个“最小核心”锤头状核酶，我们观察到假型HIV的仅弱抑制。为了评估最小核酶的重要性，我们用具有增强的自切割活性的扩展的、三级稳定的锤头状核酶代替它们，除了具有活性位点突变的非切割核酶。延伸锤头状核酶的活性和非活性版本都增加了对假型病毒的抑制，表明加工对于生物活性不是必需的。克隆稳定的细胞系表达来自这些修饰的构建体的适体强烈抑制感染性病毒，并且在高病毒感染复数（MOI）下比最小核酶更有效。三级稳定化大大增加了适体在病毒和亚细胞区室中的积累，同样与自切割能力无关。因此，我们提出，增加的积累是负责增加的抑制，适体的生物活性形式是未切割或部分切割的转录本之一，三级稳定化通过减少核酸外切酶降解来增加转录本的稳定性。
RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two "minimal core" hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation.