RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.

RNA 干扰 (RNAi) 是植物和无脊椎动物中的主要抗病毒机制，但病毒感染后哺乳动物细胞中缺乏可检测的病毒 (v) siRNAs 质疑该途径在哺乳动物免疫中的功能相关性。我们设计了一系列专门针对肠道病毒 A71 (EV-A71) 编码的蛋白 3A，一种 RNAi (VSR) 病毒抑制因子的肽。这些肽在感染细胞中废除了 EV-A71 的 VSR 功能，并导致 vsiRNA 的积累和病毒复制的减少。这些 vsiRNA 是有功能的，正如目标 RNA 的 RISC 加载和沉默所证明的那样。VSR 靶向肽 (VTP) 对 EV-A71 以及另一种肠道病毒柯萨奇病毒-A16 感染的影响在删除 RNAi 途径的核心成分 Dicer1 或 AGO2 后被消融。体内，VTP 治疗通过可检测的 vsiRNA 保护小鼠免受致命的 EV-A71 攻击。我们的研究结果为 RNAi 在哺乳动物免疫中的功能相关性提供了证据，并提出了传染病的治疗策略。