Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

Abstract

RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

“…Twenty‐four hours post‐transfection, we extracted total RNA for Northern blot analysis using a probe specific for dsRNA‐GFP. As reported (Maillard et al , 2016), dsRNA‐GFP was cleaved into siRNAs of ~22 nt in length in Ifnar1 −/− cells. Notably, we observed a dramatic reduction in small RNA production upon doxycycline‐induced LGP2 expression (Fig 5B), but not upon induction of expression of the LGP2 CTD or the CTD K634E mutant (Fig EV4C–E).…”

“…To determine the functional implications of the LGP2‐dependent block of dsRNA processing by Dicer, we used a previously established flow cytometry‐based dsRNAi reporter system that allows us to measure long dsRNA‐mediated gene silencing at a single‐cell level (Maillard et al , 2016). The Ifnar1 −/− iLGP2 cells described above stably express a destabilised form of GFP (d2GFP), allowing us to monitor GFP expression levels by flow cytometry following transfection with Cy5‐labelled dsRNA‐GFP or, as a control, Cy5‐dsRNA‐RL.…”

“…The Ifnar1 −/− iLGP2 cells described above stably express a destabilised form of GFP (d2GFP), allowing us to monitor GFP expression levels by flow cytometry following transfection with Cy5‐labelled dsRNA‐GFP or, as a control, Cy5‐dsRNA‐RL. Delivery of dsRNA‐GFP, but not dsRNA‐RL, induces a decrease in the expression of d2GFP in a Dicer‐ and Ago2‐dependent manner in these MEFs (Maillard et al , 2016). We transfected four individual Ifnar1 −/− iLGP2 clones, cultured in the absence or presence of doxycycline, with dsRNA‐RL or dsRNA‐GFP and monitored d2GFP expression 48 h later.…”

“…Those observations led to the hypothesis that the IFN system, which is attenuated in undifferentiated cells and germ cells (Pare & Sullivan, 2014), may inhibit or mask antiviral RNAi in mammalian somatic cells. In line with this hypothesis, we recently demonstrated that ablation of the IFN response by genetic removal of IFNAR or MAVS in mouse embryonic fibroblasts (MEFs) enables dsRNAi (Maillard et al , 2016). Our study, therefore, indicated that the IFN system, through induction of one or more ISGs, suppresses RNAi.…”

“…Our study, therefore, indicated that the IFN system, through induction of one or more ISGs, suppresses RNAi. This was likely happening at the level of Dicer processing, as the response to exogenous siRNAs was unaltered in IFN‐deficient versus IFN‐sufficient cells (Maillard et al , 2016). …”