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RNA viruses can hijack vertebrate microRNAs to suppress innate immunity

Abstract

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses1. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs2,3. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3′ non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

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Figure 1: EEEV restriction in myeloid cells is due to miR-142-3p binding sites in the 3′ NTR.
Figure 2: miR-142-3p binding sites in EEEV 3′ NTR decrease virus replication in the lymph node and enhance disease progression.
Figure 3: EEEV sequences containing the miR-142-3p binding sites in EEEV are required for efficient mosquito infection.

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Acknowledgements

We thank M. Dunn for excellent technical support, and M. Diamond for reading of the manuscript. This work was supported by National Institutes of Health (NIH) training grants AI049820-10 and AI060525-08 (D.W.T.), research grants AI083383 and AI095436 (W.B.K.) and a Project Grant (K.D.R.) from National Institute of Allergy and Infectious Diseases through the Pacific Northwest Regional Centers for Excellence in Biodefense and Emerging Infectious Diseases Research (U54 AI081680). The views expressed are those of the authors and do not necessarily represent the views of the NIH.

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Authors

Contributions

D.W.T., S.C.W., K.D.R. and W.B.K. designed the experiments and analysed the data. D.W.T., C.L.G., A.D.H. and E.W. performed the experiments. C.S., E.C. and A.M. provided key reagents. D.W.T. and W.B.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to William B. Klimstra.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 EEEV 3′ NTR does not restrict translation in BHK-21 fibroblasts.

a, Wild-type EEEV and wild-type VEEV translation reporters encode the translational initiation control sequences fused to the Firefly luciferase (fLuc) gene. b, The EEEV 5′Δ NTR and VEEV 5′Δ NTR encode the truncated nsP1 gene and only the 3′ NTR of either EEEV or VEEV, respectively. c, The 3′ NTR of EEEV or VEEV was inserted into a host mRNA mimic reporter to generate the 5′ host 3′ EEEV or 5′ host 3′ VEEV reporters. All translation reporters contain a 5′ cap and a 3′ poly(A) tail. d, Translation of wild-type EEEV, EEEV 5′Δ NTR, and 5′ host 3′ EEEV reporters in BHK-21 cells. Error bars represent mean ± s.d. and the data are averaged from three independent experiments performed in triplicate. WT, wild type.

Extended Data Figure 2 VEEV 3′ NTR does not restrict translation in myeloid cells.

a, b, Translation of wild-type VEEV, VEEV 5′Δ NTR, and 5′ host 3′ VEEV reporters in RAW (a) and BHK-21 (b) cells. Error bars represent mean ± s.d. and the data are averaged from three independent experiments performed in triplicate.

Extended Data Figure 3 Removal of miR-142-3p binding sites in the 3′NTR of EEEV does not alter replication in BHK-21 fibroblasts.

a, Red boxes indicate the four miR-142-3p binding sites in the 3′ NTR. Numbers represent nucleotide (nt) positions at the start and end of each miRNA binding site. b, Grey boxes correspond to the complimentary nts in the EEEV 3′ NTR and miR-142-3p. c, EEEV mutant 11337 contains a deletion in the 3′ NTR from nt 11,337 to 11,596. d, Replication of wild-type EEEV and 11337 in BHK-21 cells. n = 3 independent experiments. Error bars indicate geometric mean ± s.d., and asterisks indicate differences that are statistically significant (**P < 0.01).

Extended Data Figure 4 miR-142-3p binding sites in EEEV restrict replication in human macrophage/monocyte cell lines and primary murine Ifnar1−/− BMDCs.

a, b, Replication of wild-type EEEV and 11337 in human K562 (a) and THP-1 (b) cells. n = 2 (THP-1) and 3 (K562) independent experiments. c, Removal of type I IFN does not alleviate wild-type EEEV restriction in primary murine Ifnar1−/− BMDCs. n = three independent experiments. Data represent the geometric mean ± s.d., and asterisks indicate differences that are statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

Extended Data Figure 5 Relative expression of miR-142-3p in mouse and human cells.

Quantitative RT–PCR on primary murine BMDCs, murine and human monocyte/macrophage cell lines, and BHK-21 cells expressing miR-142-3p (BHK-21 w/miR-142-3p). Fold increase in expression is calculated compared to expression of miR-142-3p in BHK-21 cells in which miR-142-3p expression was undetectable.

Extended Data Figure 6 Specific deletion of the miR-142-3p binding sites in the 3′ NTR of wild-type EEEV does not alter replication in BHK-21 fibroblasts.

a, EEEV 142del virus contains four deletions corresponding to the complimentary nucleotides in the 3′ NTR that bind to miR-142-3p, eliminating all four miR-142-3p binding sites. b, EEEV 142pm virus contains three point mutations in each of the miR-142-3p binding sites that correspond to the seed sequence of miR-142-3p. c, Replication of wild-type EEEV and 11337 in BHK-21 cells. n = three independent experiments. Error bars indicate geometric mean ± s.d.

Extended Data Figure 7 Type I IFN attenuates 11337 and 71–77/11337.

Survival curves in Ifnar1−/− mice. n = 8 and 10 (71–77/11337) mice per virus from two independent experiments.

Extended Data Figure 8 EEEV 11337 and 71–77/11337 infect myeloid lineage cells in the PLN.

a, Per cent virus-infected cells in PLN in naïve, wild-type VEEV-, wild-type EEEV-, 71–77-, 11337- and 71–77/11337-infected mice. Plots are representative of n = 4 (naive), 5 (71–77) or 6 (71–77/11337) mice from two independent experiments. b, c, Wild-type VEEV, 11337 and 71–77/11337 infect myeloid lineage cells in the PLN. b, Representative flow plot from 1 mouse of CD11b (y-axis) and CD11c (x-axis) expression on virus-infected cells. n = 4 (naïve), 5 (71–77) or 6 (71–77/11337) mice from two independent experiments. c, Summary of CD11b and CD11c expression on virus-infected cells from wild-type VEEV-, 11337- and 71–77/11337-infected PLNs. Only mice with responses above naive mice background levels were used to determine CD11b and CD11c expression.

Extended Data Table 1 Primers used to generate the translation reporters using the QuikChange II XL mutagenesis kit
Extended Data Table 2 Primers used in the generation of EEEV mutant viruses

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Trobaugh, D., Gardner, C., Sun, C. et al. RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 506, 245–248 (2014). https://doi.org/10.1038/nature12869

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