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

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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

L.P.-O, B. M., R. P. D. and T. R. O’B. are inventors on a patent application filed by the National Cancer Institute on the basis of these findings.

Figures

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

RNA-seq in primary human hepatocytes (PHH) treated with 50 ug/ml PolyI:C for 0, 1, 2, 4, 8 or 24 hours. RNA-seq plot of the 150-Kb region in USCS browser shows expression of IFNL1, IFNL2 and IFNL3, and a novel transcribed region upstream of IFNL3. The number of reads (depth) corresponds to the level of mRNA expression. a. Detailed view of the RNA-seq results for IFNL3 and the novel transcribed region. A CTCF transcriptional insulator (ENCODE data) between these regions indicates their independence. b. Splicing architecture of the ten novel transcripts (NCBI accession numbers are presented in Supplementary Table 2). The GWAS marker, rs12979860, is located within the first intron, while a novel marker, ss469415590-TT/ΔG, is located within the first exon, common for all transcripts. Transcription and translation start sites are marked by black and blue arrows; open reading frames are shaded in blue. * -transcripts which carry premature stop codons and are likely to be eliminated by nonsense-mediated decay. Arrows indicate location of primers (Supplementary Table 1) used to generate PCR-products presented on panel d. c. PCR results in PHH cDNA using primers from panel c. No distinct PCR product is expected in the TT/TT sample; in the ΔG/ΔG sample these primers capture transcripts producing proteins of p179, 131 and 107 aa, but not of 170 aa; a transcript producing a 93 aa protein fragment is expected to be degraded. IFNL3, IFNL1 and PPIA (endogenous control) were measured in the same samples. Similar amounts of DNaseI-treated high quality RNA was used for all the reactions.

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 mature IFNL3 protein, (without leader peptide). Indicated: cysteins conserved between IFNL4 and all other IFNL proteins (C16, C50, C115, C148 and C174), interaction sites of IFNLs with their first common receptor IFNLR1 (aa 27, 33, 34, 36, 37, 44, 53, 155, 158); and interaction sites with their second receptor, IL10R2 (aa 97, 100).

Figure 3. Median decrease in HCV RNA (log10 IU/ml) in African-American participants in Virahep-C study during the first 28 days of treatment with pegIFN-α/RBV

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

Figure 4. Analysis of biological activity of 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 (mock), or treated with 10 ng/ml of recombinant purified IFN-α, IFNL3, IFNL4, or with PBS (mock). All results represent mean values of two independent biological transfection/treatment replicates, with standard deviations. b. Results of transient transfection of the IFNL4, p131 and p107 expression constructs and treatment with recombinant purified IFN-α and IFNL3 proteins in HepG2 cell line transiently co-transfected with ISRE-Luc reporter. The results are normalized to transfection with an empty vector (mock) and represent mean values of 8 biological replicates, with standard errors. c. Results of transient transfection of the IFNL4, p131 and p107 expression constructs in HepG2 cell line stably expressing the ISRE-Luc reporter. The results are normalized to transfection with an empty vector (mock) and represent mean values of 11 biological replicates, with standard errors. d. Test for antiviral effects of the IFNL4, p131 and p107 expression constructs transiently transfected into Huh7-Lunet cells stably expressing a subgenomic, luciferase-expressing HCV replicon (HCV-Luc), compared to an empty vector (mock). Results represent mean values of 4 biological replicates, with standard errors. e. Western blot analysis of Y701-STAT1 and Y689-STAT2 phosphorylation in HepG2 cells transiently transfected with expression constructs for the six protein isoforms. All Halo-tag expression constructs produce proteins detectable with an antibody for the Halo-tag; a rabbit monoclonal anti-IFNL4 antibody recognizes IFNL4 (p179) as well as its nonfunctional isoforms p131 and p107.

Figure 5. Confocal imaging of IFNL4 in primary human hepatocytes (PHH) from liver donors with different ss469415590 genotypes

Confocal imaging in PHH treated with 50 ug/ml of PolyI:C for 0, 2, 4, 8 or 24 hours. Red staining – IFNL4, green – cytoskeleton (α-tubulin), blue – nuclei. Confocal imaging of endogenous IFNL4 expression in the same ss469415590-ΔG/TT sample after in-vitro infection with JFH1-HCV strain is presented in Supplementary Fig. 6.