A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus

Journal name:
Nature Genetics
Volume:
45,
Pages:
164–171
Year published:
DOI:
doi:10.1038/ng.2521
Received
Accepted
Published online

Abstract

Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.

At a glance

Figures

  1. Identification of a novel transcribed region upstream of IFNL3.
    Figure 1: Identification of a novel transcribed region upstream of IFNL3.

    (a) RNA-seq in PHHs treated with 50 μg/ml polyI:C for 0, 1, 2, 4, 8 or 24 h. The RNA-seq plot of the 150-kb region in the USCS Genome Browser shows expression of IFNL1, IFNL2, IFNL3 and a novel transcribed region upstream of IFNL3. The number of reads (depth) corresponds to the level of mRNA expression. (b) Detailed view of the RNA-seq results for IFNL3 and the novel transcribed region. A CTCF transcriptional insulator (Encyclopedia of DNA Elements (ENCODE) data)24 between these regions indicates their independence. (c) Splicing architecture of the ten novel transcripts (NCBI accession numbers are presented in Supplementary Table 2). The GWAS marker rs12979860 is located within intron 1, and a novel marker, ss469415590, with TT or ΔG alleles is located within exon 1 and is common to all transcripts. Transcription and translation start sites are marked by black and blue arrows, respectively; ORFs are shaded in blue. Asterisks indicate transcripts that carry premature stop codons and are likely to be eliminated by nonsense-mediated mRNA decay. Arrows indicate the location of the primers (Supplementary Table 1) used to generate PCR products. aa, amino acids. (d) PCR results in PHH cDNA using the primers from c. No distinct PCR product is expected in the sample with the TT/TT genotype. In the sample with the ΔG/ΔG genotype, the primers captured transcripts producing proteins of 179, 131 and 107 amino acids but not of 170 amino acids. A transcript producing a 93-amino-acid protein fragment is expected to be degraded. IFNL3, IFNL1 and PPIA (endogenous control) levels were measured in the same samples. Similar amounts of DNase I–treated high-quality RNA were used for all reactions.

  2. Protein sequence analysis.
    Figure 2: Protein sequence analysis.

    ClustalW protein sequence alignment for IFNL4 (p179), p131, p107, IFN-λs (IFNL1, IFNL2 and IFNL3) and IFN-α. Identical amino acids are shaded in black. Exon numbering and the location of ss469415590 and other variants identified by sequencing of 270 HapMap samples are based on IFNL4 protein sequence. Annotations of protein helices, amino-acid numbering and specific amino acids are based on the sequence of the mature IFNL3 protein26, 27 (without leader peptide). Cysteines conserved between IFNL4 and all other IFN-λ proteins are indicated (at residues 16, 50, 115, 148 and 174), as are the interaction sites of IFN-λs with their first common receptor IFNLR1 (residues 27, 33, 34, 36, 37, 44, 53, 155 and 158) and with their second receptor, IL10R2 (residues 97 and 100).

  3. Median decrease in HCV RNA levels (log10 international units (IU)/ml) in African-American participants in the Virahep-C study during the first 28 d of treatment with pegIFN-[alpha]/RBV.
    Figure 3: Median decrease in HCV RNA levels (log10 international units (IU)/ml) in African-American participants in the Virahep-C study during the first 28 d of treatment with pegIFN-α/RBV.

    P = 0.015 for the mean differences in HCV RNA levels at day 28 for each of the three genotype groups at ss469415590 relative to the respective rs12979860 genotype groups.

  4. Analysis of the biological activity of the novel proteins.
    Figure 4: Analysis of the biological activity of the novel proteins.

    (a) Pathway Finder Analysis using luciferase reporter constructs representing 45 human signaling pathways in HepG2 cells. The cells were transfected with expression constructs or an empty vector or treated with 10 ng/ml recombinant purified IFN-α, IFNL3 or IFNL4 or with PBS. All results represent the mean values of two independent biological transfection and/or treatment replicates. Error bars, s.d. The red box indicates reporters significantly induced by treatment with IFN-α, IFNL3 and IFNL4. (b) Luciferase activity after transfection with vector expressing IFNL4, p131 or p107 and treatment with recombinant purified IFN-α or IFNL3 in the HepG2 cell line transiently cotransfected with the ISRE-Luc reporter. The results are normalized to the activity seen after transfection with empty vector (mock) and represent the mean values of eight biological replicates. Error bars, s.e.m. (c) Luciferase activity after transient transfection with vector expressing IFNL4, p131 or p107 in the HepG2 cell line stably expressing the ISRE-Luc reporter. The results are normalized to the activity seen after transfection with empty vector (mock) and represent the mean values of 11 biological replicates. Error bars, s.e.m. (d) Test for antiviral effects of the expression constructs for IFNL4, p131 and p107 transiently transfected into Huh7-Lunet cells stably expressing a subgenomic luciferase-expressing HCV replicon (HCV-Luc) compared to the effect seen after transfection with empty vector (mock). Results represent the mean values of four biological replicates. Error bars, s.e.m. (e) Protein blot analysis of STAT1 phosphorylated at Tyr701 (pSTAT1) and STAT2 phosphorylated at Tyr689 (pSTAT2) in HepG2 cells transiently transfected with constructs expressing the six protein isoforms. All constructs encoding the Halo tag produce proteins detectable with an antibody for Halo; the rabbit monoclonal antibody to IFNL4 recognizes p179 as well as the nonfunctional isoforms p131 and p107.

  5. Confocal imaging of IFNL4 in PHHs from liver donors with different ss469415590 genotypes.
    Figure 5: Confocal imaging of IFNL4 in PHHs from liver donors with different ss469415590 genotypes.

    Confocal imaging in PHHs treated with 50 μg/ml of polyI:C for 0, 2, 4, 8 or 24 h. Red, IFNL4; green, cytoskeleton (α-tubulin), blue, nuclei. Confocal imaging of endogenous IFNL4 expression in the same ss469415590 samples with ΔG/TT genotype after in vitro infection with the HCV JFH1 strain is presented in Supplementary Figure 6. Scale bars, 10 μm.

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Author information

Affiliations

  1. Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.

    • Ludmila Prokunina-Olsson,
    • Brian Muchmore,
    • Wei Tang,
    • Dianna Hergott,
    • Patricia Porter-Gill,
    • Adam Mumy,
    • Indu Kohaar,
    • Nathan Brand,
    • McAnthony Tarway &
    • Luyang Liu
  2. Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.

    • Ruth M Pfeiffer
  3. Immunology Section, Liver Diseases Branch, National Institute for Diabetes, Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, USA.

    • Heiyoung Park &
    • Barbara Rehermann
  4. Division of Therapeutic Proteins, Center for Drug Evaluation & Research, US Food and Drug Administration, Bethesda, Maryland, USA.

    • Harold Dickensheets,
    • Faruk Sheikh &
    • Raymond P Donnelly
  5. Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.

    • Dianna Hergott &
    • Thomas R O'Brien
  6. Information Management Services, Silver Spring, Maryland, USA.

    • Sabrina Chen
  7. Department of Epidemiology, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland, USA.

    • Jacquie Astemborski &
    • David L Thomas
  8. Department of Medicine, Carolinas Medical Center, Charlotte, North Carolina, USA.

    • Herbert L Bonkovsky
  9. National Development and Research Institutes, New York, New York, USA.

    • Brian R Edlin
  10. Center for the Study of Hepatitis C, Weill Medical College of Cornell University, New York, New York, USA.

    • Brian R Edlin
  11. Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Charles D Howell
  12. Gastroenterology Service, Veterans Affairs (VA) Long Beach Healthcare System, Long Beach, California, USA.

    • Timothy R Morgan
  13. Division of Gastroenterology, University of California–Irvine, Irvine, California, USA.

    • Timothy R Morgan
  14. Division of Infectious Diseases, Johns Hopkins University, Baltimore, Maryland, USA.

    • David L Thomas

Contributions

L.P.-O. and T.R.O. conceived and supervised the project. J.A., H.L.B., B.R.E., C.D.H., T.R.M. and D.L.T. performed the clinical and epidemiological studies from which DNA samples and data were collected. L.P.-O., B.R. and R.P.D. designed the experiments. L.P.-O., B.M., W.T., H.P., H.D., D.H., P.P.-G., I.K., A.M., N.B., M.T., L.L., F.S., B.R. and R.P.D. performed experiments and analysis. T.R.O., R.M.P. and S.C. performed statistical analysis of genetic association. L.P.-O. and T.R.O. wrote the manuscript. All authors contributed to scientific discussions and approved the final manuscript.

Competing financial interests

L.P.-O., B.M., R.P.D. and T.R.O. are inventors on a patent application filed by the National Cancer Institute on the basis of these findings.

Corresponding authors

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    Supplementary Tables 1–13, Supplementary Figures 1–11 and Supplementary Note

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