Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, USA.
Mol Ther. 2012 Apr;20(4):840-8. doi: 10.1038/mt.2012.3. Epub 2012 Jan 31.
Resistance to existing HIV therapies is an increasing problem, and alternative treatments are urgently needed. RNA interference (RNAi), an innate mechanism for sequence-specific gene silencing, can be harnessed therapeutically to treat viral infections, yet viral resistance can still emerge. Here, we demonstrate that HIV can develop indirect resistance to individual and combinatorial RNAi-targeting protein-coding regions up to 5,500 nucleotides (nt) downstream of the viral promoter. We identify several variants harboring mutations in the HIV promoter, and not within the RNAi targets, that produce more fully elongated transcripts. Furthermore, these variants are resistant to the RNAi, potentially by stoichiometrically overwhelming this cellular mechanism. Alarmingly, virus resistant to one short hairpin RNA (shRNA) also exhibits cross-resistance to a different shRNA, which targets a distinct and spatially distant region to which the virus has not been previously exposed. To our knowledge, this is the first example of HIV "cross-resistance" to viral inhibitors targeting different loci. Finally, combining anti-HIV RNAi with a small molecule enhancer of RNAi can inhibit the replication of an indirectly resistant mutant. These results suggest that indirect resistance to RNAi is a general mechanism that should be considered when investigating viral resistance and designing combinatorial RNAi therapies.
现有 HIV 治疗方法的耐药性是一个日益严重的问题，因此迫切需要替代疗法。RNA 干扰 (RNAi) 是一种针对特定序列的基因沉默的天然机制，可以被用于治疗病毒感染，但病毒仍然可能产生耐药性。在这里，我们证明 HIV 可以对个体和组合的 RNAi 靶向蛋白编码区产生间接耐药性，距离病毒启动子下游可达 5500 个核苷酸 (nt)。我们鉴定了几种携带 HIV 启动子突变而不是 RNAi 靶点突变的变体，这些变体产生了更长的转录本。此外，这些变体对 RNAi 具有抗性，可能通过化学计量比抑制这种细胞机制。令人震惊的是，对一种短发夹 RNA (shRNA) 有抗性的病毒也对另一种靶向不同且空间上不同的区域的 shRNA 具有交叉抗性，而该区域之前病毒没有暴露过。据我们所知，这是 HIV 对不同靶位的抗病毒抑制剂产生“交叉耐药性”的第一个例子。最后，将抗 HIV RNAi 与 RNAi 的小分子增强剂相结合可以抑制间接耐药突变体的复制。这些结果表明，RNAi 的间接耐药性是在研究病毒耐药性和设计组合 RNAi 疗法时应考虑的一个普遍机制。
