Enhanced RNAi does not provide efficient innate antiviral immunity in mice in vivo (ESCs small RNA-Seq)

In RNA interference (RNAi), long double-stranded RNA (dsRNA) is cleaved by Dicer endonuclease into small RNA interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates adopted sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified mouse Dicer locus to express a truncated variant (Dicer?HEL1) known to stimulate RNAi. Next, we analyzed how Dicer?HEL1/wt mice respond to four RNA viruses: Coxsackievirus B3 (CVB3) and encephalomyocarditis virus (ECMV) from Picornaviridae; tick-borne encephalitis virus (TBEV) from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in Dicer?HEL1/wt mice had no antiviral effect. This result supports insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also report that sufficiently high expression of Dicer?HEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo. Overall design: Bulk small RNA-seq of mouse embryonic stem cells (mESCs) of different Dicer and Pkr genotypes, infected with LCMV in 0.01 or 1 MOI (Multiplicity of Infection), or transfected with a plasmid expressing a MosIR dsRNA hairpin: - LCMV infection = 4x Dicer wt/wt at 0.01 MOI + 2x Dicer wt/wt at 1 MOI; 4x Dicer ?HEL1/?HEL1 at 0.01 MOI + 2x Dicer ?HEL1/?HEL1 at 1 MOI; 2x Dicer ?HEL1/?HEL1, Pkr -/- at 0.01 MOI + 2x Dicer ?HEL1/?HEL1, Pkr -/- at 1 MOI; 2x Dicer -/- at 0.01 MOI; 4x Dicer S-4 at 0.01 MOI; 4x Dicer O-3 at 0.01 MOI - MosIR transfection = 2x Dicer wt/wt; 2x Dicer ?HEL1/?HEL1; 2x Dicer S-4; 2x Dicer O-3