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.