In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system1,2. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system2. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.
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A-to-I editing of Malacoherpesviridae RNAs supports the antiviral role of ADAR1 in mollusks
BMC Evolutionary Biology Open Access 23 July 2019
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We thank J. K. Lim and A. G. Pletnev for Langat virus reagents, B. Lee for Sendai virus reagents, R. W. Hardy for SINV reagents, B. R. Cullen for NoDice cells, K. K. Conzelmann for BSR-T7 cells, and B. Ramratnam for pEGFP–Drosha. Recombinant IFN-β was provided by the National Institute of Health’s Biodefense and Emerging Infections Research Resources Repository (HuIFN-β, NR-3080). This material is based upon work supported in part by the Burroughs Wellcome Fund, which provides support for both S.C. and B.R.T. S.C. is also supported by the National Institute of Allergy and Infectious Diseases (NIAID) (R01A1074951). J.P.L. is supported by the DIM Malinf, Conseil Regional d’Ile-de-France. L.C.A. is partly supported by the American Heart Association (15PRE24930012). A.E.S. is supported by National Science Foundation (MCB-1411836) and NIAID (R21AI117882). J.M. is supported by National Institute of General Medicine (F32 GM119235). B.R.T. is also supported by NIAID (R01AI110575).
The authors declare no competing financial interests.
Reviewer Information Nature thanks B. Cullen, L. Maraffini and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Figure 1 Characterization of RNaseIII−/− cells.
a, Sequence alignment of genetic alterations in the two alleles encoding Drosha in RNaseIII−/− cells. The deletion and insertion result in a frameshift and early stop codon. b, The ten most abundant miRNAs in each condition, parental HEK293T (WT) or RNaseIII−/− either mock-treated or SINV-infected for 24 h as determined by Illumina small RNA deep sequencing. c, Quantitative PCR (qPCR) analysis of DGCR8 mRNA levels in mock-treated and SINV-infected (MOI = 0.1, 8 h.p.i.) NoDice and RNaseIII−/− cells; error bars, s.d. from three independent experiments; all conditions had P < 0.05 by Student’s t-test. d, Transcriptome profiling and correlation analyses of NoDice cells at baseline and RNaseIII−/− cells transfected with GFP-tagged human Drosha for 72 h. Graph depicts data from two biological replicates per condition.
Extended Data Figure 2 Drosha depletion does not alter the response to IFN-I.
a, qPCR analysis of IFIT1 mRNA levels in NoDice and RNaseIII−/− fibroblasts treated with IFN-β (100 U ml−1, 8 h); error bars, s.d. from three independent experiments; all conditions had P < 0.05 by Student’s t-test. b, Western blot of NoDice and RNaseIII−/− fibroblasts infected with SINV for 1 h before administration of indicated amounts of IFN-β for 24 h. c, d, Northern blot (c) and western blot (d) of primary Rnasenf/f ear-derived fibroblasts treated with indicated AdVs for 2–3 days and then treated with either 100 U IFN-β for 6 h (c) or infected with SINV for 24 h (d). e, mRNA sequencing of total RNA from samples in c. Heatmap depicts known mouse ISGs and IFN downregulated genes with a log2(fold change) greater than 1, as defined by the Interferome database (www.interferome.org).
Extended Data Figure 3 Characterization of Drosha-2A cells.
a, Immunofluorescence of human fibroblasts stably expressing GFP-tagged Drosha-WT or Drosha-2A (S300A/S302A). b, The indicated Flag-tagged proteins were immunoprecipitated from whole-cell extracts (WCE) and incubated at 37 °C with in vitro transcribed genome of SIN124. Production of pre-miR-124 was determined by small RNA northern blot. c, Indicated cell types were infected with SINV at an MOI of 0.001 and viral titres were determined at 16 h.p.i. Shown is the average and s.d. of three independent experiments, with P < 0.05 as determined using a two-tailed Student’s t-test.
Extended Data Figure 4 Drosha-RB-RIIIDmut recognizes stem-loop structures in SINV RNA.
a, Immunoprecipitation of exogenously expressed Flag-tagged proteins. Shown is protein expression in the whole-cell extract and after immunoprecipitation (IP) with a Flag-specific antibody. b, Cells were transfected with Flag-tagged SeV-N or Drosha-RBmut and infected at 36 h.p.t. with SINV at an MOI of 3. At 8 h.p.i., Flag-tagged proteins were immunoprecipitated and bound RNA was isolated to perform qPCR. Graph shows SINV RNA levels relative to input and normalized to tubulin. The average of three independent experiments is shown. Error bars, s.d.; *P < 0.05 using a one-tailed Student’s t-test. c, Prediction of the structure of the 5′ 200 nucleotides of the SINV genome using RNAfold. d, EMSA was performed with the indicated immunoprecipitated proteins and radio-labelled in vitro transcribed RNA comprising the 5′ 200 nucleotides of the SINV genome. Unbound genome is indicated as ‘Free RNA’.
Extended Data Figure 5 Using virus engineering to discern Drosha’s antiviral mechanism.
a, Schematic of the SINV replicon encoding Gaussia luciferase in place of the structural polyprotein used in Fig. 3e–g. b, Schematic of the SINV temperature-sensitive mutant (SIN-RdRpts). Star denotes ts point mutant. c, NoDice and RNaseIII−/− cells were infected with virus depicted in b, at an MOI of 10 and incubated at 40 °C, a temperature at which the mutant viral RdRp is completely inactive. Levels of genomic (g) SINV RNA were determined by qPCR at the indicated times after infection. Data are representative of two independent experiments where each condition was done in triplicate; error bars, s.d. d, Schematic of the SINV encoding firefly luciferase in the nsP3 region and an inactive RdRp (SIN-nsP3Luc) e, Graph depicts levels of in vitro translation of firefly luciferase produced from virus in d, in the presence of membrane fractions from control or Drosha-2A. The data shown are the average of three independent experiments; error bars, s.d.
Extended Data Figure 6 Localization of and miRNA production from cytoplasmic viruses in diverse eukaryotes.
a, Zebrafish embryos were inoculated with SINV for 24 h and then analysed by FISH using a probe complementary to the capsid region of the genome. b, qPCR analysis of zebrafish embryos treated with the indicated morpholinos for 2 days (n = 4); error bars, s.d. c, A. thaliana protoplasts were mock-treated or TCV-infected for 40 h and then analysed by FISH using a Cy3-labelled probe complementary to bases 1210–1259 of the TCV genome. d, Quantification of mature miR-124 production from recombinant TCV was performed using the TaqMan miRNA assay on RNA from Fig. 4b. All samples were normalized to endogenous snoR66. Quantifications of each sample were performed in triplicate; error bars, s.d. from two biological replicates.
Extended Data Figure 7 The impact of diverse RNase III members on virus infection.
a, Schematic depicting core domains of human Drosha, C-terminal region of C. intestinalis Drosha, or full-length RNase III of S. pombe, M. maripaludis, and S. pyogenes. Domains depicted include Proline-rich (P-rich), arginine–serine-rich (RS-rich), conserved central domain (CED), RNaseIII domain (RIIID), and double-stranded RNA binding domain (dsRBD). b, Western blots from BSR-T7 cells, co-transfected with the indicated RNase-III-expression plasmids and SeV rescue plasmids encoding SeV-GFP genome, SeV-N, SeV-P, and SeV-L genes. RNase III expression was determined at 48 h.p.t. and virus replication at 72 h.p.t. c, Western blot of DL1 cells treated with indicated dsRNA for 3 days and subsequently infected with SINV (MOI = 1) for 96 h. d, HEK293T (WT) or RNaseIII−/− cells were infected with SINV for 24 h. Graphs depict the number of SINV reads mapping to indicated positions along the viral genomes from the small RNA deep sequencing performed in Extended Data Fig. 1b.
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Aguado, L., Schmid, S., May, J. et al. RNase III nucleases from diverse kingdoms serve as antiviral effectors. Nature 547, 114–117 (2017). https://doi.org/10.1038/nature22990
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