Long dsRNA mediated RNA interference (dsRNAi) is antiviral in interferon competent mammalian cells

Abstract In invertebrate cells, RNA interference (RNAi) acts as a powerful defense against virus infection by cleaving virally produced long dsRNA into siRNA by Dicer and loaded into RISC which can then destroy/disrupt complementary viral mRNA sequences. Comparatively in mammalian cells, the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. Although the cellular machinery for RNAi functions in mammalian cells, its role in the antiviral response remains controversial. Here we show that IFN competent mammalian cells engage in dsRNA-mediated RNAi. We found that pre-soaking mammalian cells with concentrations of sequence-specific dsRNA too low to induce IFN production could significantly inhibit viral replication, including SARS-CoV-2. This phenomenon was dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, Dicer-knockout cell lines were incapable of this inhibition, confirming use of RNAi. This represents the first evidence that soaking with gene-specific dsRNA can generate viral knockdown in mammalian cells. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform.