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The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus–induced microRNAs

Nature Immunology volume 15, pages 72–79 (2014)Cite this article

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Abstract

IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3′ untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.

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Figure 1: The rs4803217 3′ UTR variants of IFNL3 are regulated differently and are subject to AMD.
Figure 2: Variation in the 3′ UTR of IFNL3 mediates post-transcriptional repression by the HCV-induced myomiRs miR-208b and miR-499a-5p.
Figure 3: Inhibition of HCV-induced myomiRs increases the expression of IFNL2 and IFNL3 as well as the antiviral response.
Figure 4: Induction of MYH7 and MYH7B is specific to HCV infection.
Figure 5: Expression of myosin and myomiRs in patients chronically infected with HCV.

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Acknowledgements

We thank P. Fink, D. Stetson, E. Clark and C. Lim for critical reading of the manuscript, and S. Badil and M. Hong for technical assistance. Supported by the Department of Immunology of the University of Washington (R.S.), the US National Institutes of Health (AI060389 and AI88778 to M.G., and CA148068 to C.H.H.) and federal funds from the Frederick National Laboratory for Cancer Research of the US National Institutes of Health (HHSN261200800001E) and the Intramural Research Program of the US National Institutes of Health, Frederick National Laboratory for Cancer Research and the National Cancer Institute, Center for Cancer Research (E.B., B.A.S. and M.C.).

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Author notes

  1. Stacy M Horner & Curt H Hagedorn

    Present address: Present addresses: Department of Molecular Genetics and Microbiology, Center for Virology, Duke University Medical Center, Durham, North Carolina, USA (S.M.H.), and Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA (C.H.H.).,

Authors and Affiliations

  1. Department of Immunology, University of Washington, Seattle, Washington, USA

    Adelle P McFarland, Stacy M Horner, Abigail Jarret, Rochelle C Joslyn, Michael Gale Jr & Ram Savan

  2. Basic Science Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

    Eckart Bindewald

  3. Center for Cancer Research Nanobiology Program, National Cancer Institute, Frederick, Maryland, USA

    Bruce A Shapiro

  4. Divison of Gastroenterology, Hepatology and Nutrition, School of Medicine, University of Utah, Salt Lake City, Utah, USA

    Don A Delker & Curt H Hagedorn

  5. Cancer and Inflammation Program, Laboratory of Experimental Immunology, Science Applications International Corporation–Frederick, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

    Mary Carrington

  6. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, Massachusetts, USA

    Mary Carrington

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Contributions

A.P.M. and R.S. designed the study and wrote the manuscript; R.S. directed the study; A.P.M. analyzed the data; A.P.M., A.J., R.S. and R.C.J. did ARE and miRNA experiments; S.M.H. did infections and flow cytometry preparations; E.B. and B.A.S. generated base-pairing probabilities; D.A.D. and C.H.H. contributed clinical samples; and M.C. and M.G. provided intellectual input.

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Correspondence to Ram Savan.

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Integrated supplementary information

Supplementary Figure 1 IFNL full-length 3′ UTR alignments and base pair probabilities.

(a) Alignment of full-length IFNL3-T, IFNL3-G and IFNL2 3′ UTRs. The rs4803217 SNP in the IFNL3 3′ UTR is indicated with an arrow, AREs are boxed, and the myomiR target region (discussed in Fig. 2) is highlighted in blue. (b) Schematics of the luciferase reporter constructs. Full-length IFNL3 3′ UTRs containing a T or G at nt position 53 were cloned downstream of the luciferase gene (LUC) in the pGL3 construct with an SV40 promoter. Control construct contained a minimal 3′ UTR. (c) X-axis: Position on IFNL3 3′ UTR; Y-axis: Probability of base pairing. Black: IFNL3-T 3′ UTR; Red: IFNL3-G 3′ UTR. The shown values are the position-wise sums of base pair probabilities generated by RNAfold.

Supplementary Figure 2 Additional analyses of IFNL 3′ UTRs.

(a) Alignment of full-length IFNL3-T and IFNL1 3′ UTRs. AU-rich elements (ARE) are boxed. (b) Species alignment of the region around IFNL3 3′ UTR rs4803217 (1000 Genomes release 13 - December 2012; http://browser.1000genomes.org). (c) Alignment of IFNL3 and IFNL2 3′ UTR sequences (nt 35 to 65). A T→G mutation (ΔT→G) was introduced into the 3′ UTR of IFNL2 to mimic the rs4803217 SNP present in the IFNL3 3′ UTR. (d) Luciferase expression of IFNL2 wt versus ΔARE, ***P<0.0001. Data represent six replicates in each experimental group plotted as mean ± s.e.m. from one representative of three or more experiments. An unpaired t test was used for statistical comparison and a two-tailed P value is given.

Supplementary Figure 3 myomiR regulation of IFNL2 and IFNL3 3′ UTRs.

(a) HCV viral RNA detected in Huh7 samples presented in Fig. 2c and 2d. PI, post infection. Standard curve was generated using HCV viral RNA diluted over six logs. (b, c) HepG2 cells were co-transfected with the indicated 3′ UTR luciferase reporter constructs along with miR-208b, miR-499a-5p or NC (negative control) mimics. (b) miR-208b and miR-499a-5p mimics significantly repressed the IFNL3-T 3′ UTR luciferase activity compared to NC mimics. (c) Increasing concentrations of myomiR mimics (cocktail of miR-208b+miR-499a-5p) of 5 nM, 10 nM and 20 nM were used. (b, c) Data represent six replicates in each experimental group plotted as mean ± s.e.m. from one representative of three experiments. Unpaired t tests were used for statistical comparisons and two-tailed P values are given. (d, e) HepG2 cells were treated with Poly(I:C) (d) or HCV PAMP (e) for the indicated times. Data represent mean ± s.e.m. for one of multiple comparable experiments. (f) HepG2 cells were co-transfected with HCV PAMP and NC mimics or a cocktail of miR-208b and miR-499a-5p mimics. Cells were lysed and Ago2 was immunoprecipitated (IP). Shown is an immunoblot (IB) analysis for Ago2 protein in an IgG isotype control IP and two Ago2 IPs performed on NC mimic or myomiR mimic-transfected cells. Data represent mean ± s.e.m. for one of multiple comparable experiments. Unpaired t tests were used for statistical comparisons and two-tailed P values are given. *P<0.05, **P<0.01, ***P<0.001.

Supplementary Figure 4 Analysis of IFNL 3′ UTR variants in a co-expression system.

(a) The luciferase gene of the constructs described earlier (Fig. 1b and Supplementary Fig. 1b) was swapped for an eGFP or mCherry gene. eGFP is upstream of the IFNL3-G 3′ UTR, while mCherry is upstream of the IFNL3-T 3′ UTR. (b) FACS analysis of Huh7 cells transfected with individual control plasmids or a 1:1 mixture. Experiments using a 1:1 mixture of the IFNL3 3′ UTR reporter plasmids were compared against mixtures containing a 1:1 ratio of eGFP and mCherry reporters with control 3′ UTRs.

Supplementary Figure 5 Myosin induction by HCV is RIG-I independent and is specific to HCV.

(a) HCV viral RNA detected in Huh7.5 samples presented in Figure 4a. (b) IFNB expression was assessed in SenV or WNV time courses (see Fig. 4c) compared to a mock (M) infection (time point 48 h). Data represent mean ± s.e.m. for one of two comparable experiments.

Supplementary Figure 6 Model of regulation of IFNL2 and IFNL3 by HCV-induced myomiRs.

Infection of hepatocytes by HCV directly results in cleavage of MAVS by the HCV protease NS3/4A, which impairs induction of IFNB. HCV indirectly represses IFNL2 and IFNL3 by inducing host myosin genes, MYH7 and MYH7B, that encode the myomiRs miR-208b and miR-499a-5p. These myomiRs destabilize IFNL2 and IFNL3-T (rs4803217 T variant) mRNA by targeting the 3′ UTRs, while the IFNL3-G variant escapes myomiR-mediated repression. Post-transcriptional regulation by ARE-binding proteins adds another layer of complexity, which, combined with myomiR-mediated repression, dictates the level of mRNA degradation.

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McFarland, A., Horner, S., Jarret, A. et al. The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus–induced microRNAs. Nat Immunol 15, 72–79 (2014). https://doi.org/10.1038/ni.2758

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