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.2013 Oct 11;342(6155):235-8.
doi: 10.1126/science.1241930.

Antiviral RNA interference in mammalian cells

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Antiviral RNA interference in mammalian cells

P V Maillard et al. Science..

Abstract

In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

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Figures

Fig. 1
Fig. 1. EMCV-derived siRNAs in infected mESCs
(A) Western analysis of VP1 in E14 mESCs. NI, noninfected; ACTIN, protein-loading control. (B) Size distribution of vsRNA reads mapping to EMCV in samples from (A). (C) Distributions of 21- to 23-nt and 24- to 44-nt reads along EMCV (+) and (−) strands 6 hpi. (D) Same as (C), but along the first 5′-terminal 300 nt. Symmetrical reads are numbered. (Inset) Perfect duplexes formed by the abundant reads 1 to 2 and 3 to 4; 2-nt 3′ overhangs are indicated in red. Asterisk: Read sequence corresponding to the oligonucleotide probe used in (E). Radar plots: 21- and 23-nt reads in each of 22 possible registers mapping along the entire EMCV (+) and (−) strands; the 5′ first EMCV nucleotide defines register no. 1. Distance to the center indicates read percentage within each register. (E) Northern analysis of EMCV 5′-end siRNAs 6 hpi. Total RNA from SUC-SUL (SS) transgenicArabidopsis run in parallel and hybridized secondarily provides a size marker for 21-nt and 24-nt siRNAs. U6, RNA-loading controls.
Fig. 2
Fig. 2. AGO2-loaded EMCV siRNAs are reduced inDcr−/− mESCs and following differentiation
(A) Western and Northern analysis of VP1 (top), EMCV 5′-end siRNAs (middle) and miR-16/pre-miR-16 (bottom) inDcrFlx/Flx andDcr−/− mESCs infected (+) or not (−) with EMCV. SS, as in Fig. 1E. ACTIN and U6: protein- and RNA-loading controls. (B) Northern analysis of EMCV 5′-end siRNAs 6 hpi in FLAG-specific immunoprecipitates isolated from WT mESCs or mESCs stably expressing human FLAG-HA hAGO2, infected (+) or not (−) with EMCV. SS as in (A). Western analyses show comparable infection levels (VP1) and confirm FHA-hAGO2 immunoprecipitation with miR-16. Total: Coomassie-stained protein loading control. (C) Distributions of 21- and 23-nt reads along EMCV (+) and (−) strands after deep-sequencing of RNA from endogenous mAGO2 IP 6 hpi. Asterisks: reads further analyzed in fig. S2E. (D) Same as (C), but along the first 5′-terminal 150 nt. (E) Western and Northern analyses of the pluripotency markers OCT4, VP1, EMCV 5′-end siRNAs and miR-16 in undifferentiated mESCs on day 0 (d0) or after 10 days of differentiation (d10), infected (+) or not (−) with EMCV. Total as in (B); U6 and SS are as above. (F) Size distribution of vsRNA deep-sequencing reads mapping to EMCV in samples from (E). Abundance was normalized to the total number of reads mapping to EMCV. (G) Reads mapping to EMCV in infected day 0 and day 10 mESCs, as in Fig. 1D. Note the scale change in counts, highlighted in red. (Inset) siRNA duplex 1 to 2 remains detectable in day 10 cells.
Fig. 3
Fig. 3. B2 antagonizes NoV-derived siRNA production
(A) Northern analysis of genomic RNA1 and subgenomic (sg)RNA3 in mESCs 72 hpi with NoV or NoVΔB2. Ribosomal RNA (rRNA) indicates rRNA ethidium bromide staining; NI, noninfected.(B)Normalized size distribution of deep-sequencing reads mapping the NoV or NoVΔB2 genome in samples from (A). (C) Distributions of 21- to 23-nt and 24- to 44-nt reads along the (+) and (−) strands of NoV (left) or NoVΔB2 (right) RNA1. (D) Same as (C), but along the first 5′-terminal 300 nt. (E) Radar plots as in Fig. 1D, but for NoV and NoVΔB2; RNA1 5′ first nucleotide defines register no. 1. (F) Read sequences along the first 180 nt of the 5′-terminus of NoVΔB2 RNA1 (+) and (−) strands. Read counts (in bold italic), genomic position, and sequence variants are indicated. Nonsequenced reads within the main ~22-nt vsRNA periodicity register are indicated with XXX. Reads detected identically in NoVΔB2-infected BHK-21 cells and suckling mice (23) are depicted in blue. 2-nt3′ overhangs are in red.
Fig. 4
Fig. 4. Rescue of NoVΔB2 accumulation in AGO2-deficient mESCs
(A) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of the hAgo2 transgene mRNA levels in noninfected (NI) and NoV- and NoVΔB2-infected E7 mESCs previously treated (+) or not (−) with tamoxifen for 5 days. Results show the mean and standard deviation of two independent experiments; a.u., arbitrary units. (B) Relative accumulation of NoV or NoVΔB2 RNA1 72 hpi in E7 mESC treated (+T) or not with tamoxifen, assessed by quantitative RT-PCR on samples used in (A). Results show the mean of the ratio and the standard deviation calculated from two independent experiments. (C) Northern analysis of NoV and NoVΔB2 genomic RNA1 and sgRNA3 72 hpi of the cells used in (A). rRNA, as in Fig. 3A.
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