Antiviral RNAi Restricts DNA Virus Infection in Mammals

Antiviral RNA interference (RNAi) is an established component of mammalian innate immunity, primarily recognized for targeting RNA viruses. However, whether mammalian RNAi can restrict DNA virus infection remains unclear. In this study, we uncover that double-stranded DNA (dsDNA) viruses—poxviruses, including Vaccinia virus (VACV) and Monkeypox virus (MPXV), trigger antiviral RNAi in mammalian cells. During poxvirus infection, viral dsRNAs are derived from bidirectional transcription of the viral DNA genome and elicit Dicer-dependent production of viral siRNAs (vsiRNAs), which predominantly locate to the inverted terminal repeat (ITR) regions at the termini of the poxvirus genome. Depletion of Dicer or mutagenic inactivation of AGO2’s RNA slicing activity enhances the replication of VACV and MPXV. Moreover, sequestration of ITR-derived vsiRNAs by sponge decoys increases VACV replication, attributable to the role of these vsiRNAs in inhibiting the resolution of viral concatemeric DNA genomes into mature monomers. Furthermore, AGO2-deficient cells exhibit more sensitivity to VACV infection compared to STING- or MAVS-deficient cells, suggesting that RNAi is the primary antiviral response in restricting VACV infection in mammalian cells. In VACV-infected mice, Dicer conditional knockout exacerbates disease severity, manifested by increased mortality, exacerbated pulmonary pathology and elevated viral loads. Additionally, treatment with enoxacin, an RNAi enhancer, suppresses VACV replication in an RNAi-dependent manner both in vitro and in vivo. Together, our findings provide evidence for the functional relevance of RNAi in counteracting DNA viruses in mammals and discover an unprecedented role of vsiRNAs involved in poxvirus genome replication.

抗病毒RNA干扰（RNAi）是哺乳动物先天免疫中公认的组成部分，其主要功能为靶向RNA病毒。然而，哺乳动物RNAi是否能够限制DNA病毒感染，目前仍不明确。本研究发现，双链DNA（dsDNA）病毒——包括痘苗病毒（VACV）与猴痘病毒（MPXV）在内的痘病毒——可在哺乳动物细胞中诱导抗病毒RNAi。在痘病毒感染过程中，病毒双链RNA源自病毒DNA基因组的双向转录，并经Dicer依赖的途径生成病毒小干扰RNA（vsiRNAs），这类vsiRNAs主要定位于痘病毒基因组末端的反向末端重复序列（ITR）区域。敲低Dicer或诱变灭活AGO2的RNA剪切活性，均可增强痘苗病毒与猴痘病毒的复制能力。此外，通过RNA海绵诱饵捕获ITR来源的vsiRNAs，可促进痘苗病毒的复制，这一现象归因于此类vsiRNAs能够抑制病毒串联DNA基因组裂解为成熟单体的过程。与STING缺陷型或MAVS缺陷型细胞相比，AGO2缺陷型细胞对痘苗病毒感染的敏感性更高，这表明RNAi是哺乳动物细胞中限制痘苗病毒感染的主要抗病毒应答通路。在感染痘苗病毒的小鼠体内，条件性敲除Dicer会加剧疾病严重程度，表现为死亡率升高、肺部病理损伤加重以及病毒载量上升。此外，恩诺沙星——一种RNAi增强剂——可通过RNAi依赖的方式，在体外与体内均抑制痘苗病毒的复制。综上，本研究的发现证实了RNAi在哺乳动物体内拮抗DNA病毒的功能相关性，并揭示了vsiRNAs在痘病毒基因组复制中前所未有的作用。