Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Retrovirology. 2010 Jun 8;7:52. doi: 10.1186/1742-4690-7-52.
RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence. For durable inhibition of HIV-1 replication the emergence of such escape viruses must be controlled. Here we present a strategy that anticipates HIV-1 escape by designing 2nd generation short hairpin RNAs (shRNAs) that form a complete match with the viral escape sequences.
To block the two favorite viral escape routes observed when the HIV-1 integrase gene sequence is targeted, the original shRNA inhibitor was combined with two 2nd generation shRNAs in a single lentiviral expression vector. We demonstrate in long-term viral challenge experiments that the two dominant viral escape routes were effectively blocked. Eventually, virus breakthrough did however occur, but HIV-1 evolution was skewed and forced to use new escape routes.
These results demonstrate the power of the 2nd generation RNAi concept. Popular viral escape routes are blocked by the 2nd generation RNAi strategy. As a consequence viral evolution was skewed leading to new escape routes. These results are of importance for a deeper understanding of HIV-1 evolution under RNAi pressure.
RNA 干扰（RNAi）是一种进化上保守的基因沉默机制，介导靶 mRNA 的序列特异性降解。RNAi 可以通过靶向病毒 RNA 基因组来抑制 HIV-1 的复制。然而，HIV-1 易错复制机制可以产生具有靶序列特定突变的 RNAi 抗性变体。为了持久抑制 HIV-1 的复制，必须控制此类逃逸病毒的出现。在这里，我们提出了一种通过设计与病毒逃逸序列完全匹配的第二代短发夹 RNA（shRNA）来预测 HIV-1 逃逸的策略。
为了阻断靶向 HIV-1 整合酶基因序列时观察到的两种最受欢迎的病毒逃逸途径，我们将原始 shRNA 抑制剂与两个第二代 shRNA 组合在单个慢病毒表达载体中。我们在长期病毒挑战实验中证明，两种主要的病毒逃逸途径都被有效地阻断了。然而，最终还是发生了病毒突破，但 HIV-1 的进化被扭曲，被迫使用新的逃逸途径。
这些结果证明了第二代 RNAi 概念的强大。第二代 RNAi 策略阻断了常见的病毒逃逸途径。因此，病毒进化被扭曲，导致新的逃逸途径。这些结果对于深入了解 HIV-1 在 RNAi 压力下的进化具有重要意义。
