Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells

Abstract

Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

Keywords: RNA interference; RNA virus; innate immunity.

FIG 1

Functional validation of RIG-I- and MDA5-deficient cell line generated by CRISPR-Cas9 technology. (A) Schematic representation of the RIG-I and MDA5 domain structure, the guide RNAs, and the Sanger sequence obtained. CTD, C-terminal domain. (B) Western blotting for RIG-I, MDA5, and tubulin as a loading control. Control and RIG-I- and MDA5-deficient (KO) cells were mock treated or stimulated with 200 ng poly(I⋅C) for 24 h. See Fig. S1 in the supplemental material for the complete image. (C) Control and KO cells were treated with 200 ng of poly(I⋅C) or mock treated for 24 h, and the induction of IFN-β, ISG15, and OAS1 mRNA was measured by RT-qPCR. (D) Control and KO cells were infected with yellow fever virus 17D (YFV17D) at an MOI of 0.1, and the induction of IFN-β mRNA was measured by RT-qPCR over time (hpi, hours postinfection). Data are presented as means and standard deviations from biological triplicates. One representative experiment of three independent repeats is shown. Statistical significance was determined using Student’s t test (***, P < 0.001; **, P < 0.01; *, P < 0.05).

FIG 2

Size profile of viral small RNAs obtained from control cells and RIG-I- and MDA5-deficient (KO) cells after infection for 24 h with SINV at an MOI of 0.1, for 48 h with YFV17D at an MOI of 0.1, or for 16 h with CBV3 at an MOI of 0.01. Small RNAs were mapped to the sense (blue bars) and antisense (red bars) strands of the viral genome, allowing 1 mismatch. Sense and antisense reads are presented on different scales, as antisense reads were very scarce. RPM, reads per million.