Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot
Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot
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DOI：
10.1371/journal.pone.0146710
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发表时间：
2016-01-11
期刊：
影响因子：
3.7
通讯作者：
Oh, Jong-Won
中科院分区：
文献类型：
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作者：
Moon, Jae-Su;Lee, Seung-Hoon;Oh, Jong-Won
丙型肝炎病毒(HCV)内部核糖体进入位点(IRES)是以小干扰RNA(SiRNA)为基础的抗病毒治疗的主要靶点。然而，考虑到丙型肝炎病毒IRES的二级和三级结构的复杂性以及与多种蛋白质的相互作用，基于生物信息学的siRNA设计是一项具有挑战性的任务，它还可以动态地改变这种顺式作用的RNA元件的结构。在这项工作中，我们利用siRNA平铺方法，将siRNA与重叠序列平铺，这些重叠序列在丙型肝炎病毒IRES茎环结构III和IV上移动了一个或两个核苷酸，跨越核苷酸(NTS)277-343。根据它们的抗病毒活性，我们绘制了一个可用药区域(NTS 313-343)，在那里可以浓缩有效siRNA的靶标。SiIE22显示出最强的抗丙型肝炎病毒效力，它针对不同的丙型肝炎病毒基因的高度保守的序列，位于IRES亚域IIIF内，参与假结的形成。靶向50或30方向逐级移动1或2个核苷酸降低了siIE22的抗病毒效力，表明了siRNA可访问这个高度结构和序列保守的区域对RNA干扰的重要性。纳米颗粒介导的系统递送稳定性增强的siIE22衍生物GS_PS1 siIE22，其导引链上含有单一的硫代磷酸连接，在用于丙型肝炎病毒复制的异种小鼠模型中，将血清丙型肝炎病毒基因组滴度降低了4log(10)以上，而没有产生抗药性变异。我们的结果为识别针对高结构RNA靶标的有效siRNA物种提供了一种策略，并提供了一种潜在的泛丙型肝炎病毒基因型式siRNA疗法，该疗法可能对当前治疗方案耐药的患者有利。
The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 50 or 30 direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log(10) in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.