Cave Eleanor, Weinberg Marc S, Cilliers Tonie, Carmona Sergio, Morris Lynn, Arbuthnot Patrick
HBV Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Wits 2050, Johannesburg, South Africa.
AIDS Res Hum Retroviruses. 2006 May;22(5):401-10. doi: 10.1089/aid.2006.22.401.
Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.
通过激活RNA干扰（RNAi）途径发现序列特异性沉默，为治疗人类免疫缺陷病毒1型（HIV-1）感染带来了令人兴奋的新策略。在HIV-1亚型中，C型在受影响最严重的世界地区尤为常见。尽管易于发生突变，但该亚型的基因组可塑性在功能重要区域是有限的。我们在HIV-1 C亚型gag开放阅读框内鉴定了保守序列，并评估它们是否是RNA聚合酶III驱动的小发夹RNA（shRNA）抑制病毒复制的合适靶点。最初，使用报告基因检测法确定了一组10种shRNA对HIV-1的有效性。靶向gag 5'端的shRNA A和B最有效，用于评估对两种R5分离株（Du151和Du422）和一种X4病毒（SW7）在培养细胞中的复制抑制作用。这些shRNA可将细胞内HIV-1 gag RNA和HIV-1蛋白浓度以及p24分泌减少多达80%，且不会诱导干扰素反应。然而，shRNA介导的针对每种病毒分离株的敲低效果略有不同。这些数据表明成功激活RNAi以抑制生物学上不同的HIV-1 C亚型分离株的复制。本文所述的效应shRNA是针对全球最常见的HIV-1亚型进行基因治疗应用的潜在候选物。
