Prokunina-Olsson L, Muchmore B, Tang W, Pfeiffer RM, Park H, Dickensheets H, et al. A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus. Nat Genet 2013;45:164-171. www.nature.com (Reprinted with permission.)
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.
Interferons alpha (IFN-β) and lambda (IFNL) are cytokines with key roles in combating viral infections. Engagement of IFN receptor complexes results in JAK/STAT (Janus kinase / signal transducers and activators of transcription) signaling that induces the expression of hundreds of interferon-stimulated genes (ISGs) as part of an elaborate host cell defense program adapted to combat infections.1 Pegylated IFN-β (PEG-IFN-β) in combination with ribavirin is part of the standard-of-care (SOC) treatment for chronic hepatitis C (CHC). However, treatment response is variable and dependent on several host factors including gender and race.2 For example, SOC therapy is less effective in treating African Americans (AA) than Caucasian Americans.3 Furthermore, several studies demonstrated that patients with delayed or nonresponse to IFN-β treatment have higher expression levels of ISGs prior to therapy than those successfully treated.4–6 This is likely due to a preactivated refractory state of the IFN signaling pathway.7 Previous genome-wide association studies of CHC patients have identified single nucleotide polymorphisms (SNPs) in the region of the IFNL3 (IL28B, IFN-l3) gene on chromosome 19q13.13 that correlate with both spontaneous,8, 9 and IFN-mediated HCV clearance,9–12 and could act as a predictive biomarker for SOC efficacy.13 Furthermore, other markers have been proposed to play a role in viral clearance.14, 15 One of these SNPs in the IFNL3 region at position 12979860 (rs12979860) has been linked both to hepatic ISG expression and outcome of IFN therapy for CHC.16 Although many studies have investigated the functional role of this SNP, its associated pathogenetic mechanisms remain poorly understood.17
In this context, a recent multicenter study by Prokunina-Olsson et al. identified a novel SNP at position 469415590 (ss469415590 (TT or δG)) upstream of the IFNL3 gene with potential relevance for viral pathogenesis and treatment response.18 Using viral RNA mimics with messenger RNA (mRNA) sequencing in primary human hepatocytes, the authors identified a novel region that is transiently transcriptionally activated. Interestingly, this region included a novel SNP (ss469415590[δG]), resulting in a frame shift mutation leading to the production of five transcripts, although one of these is likely eliminated by nonsense-mediated mRNA decay (Fig. 1). Genetic analysis of this region in patients from several independent clinical cohorts of hepatitis C studies revealed an association of the ss469415590[δG] allele with reduced response rates to IFN-β treatment (Virahep-C, HALT-C) and reduced association with spontaneous HCV clearance (UHS, ALIVE). The association pattern of ss469415590 to IFN-β treatment response and viral clearance was similar to rs12979860 in participants of the HALT-C and UHS trials but slightly more pronounced for ss469415590 in AA participants. Furthermore, the team demonstrated that ss469415590 is in high linkage disequilibrium with rs12979860 in the IFNL3 gene, suggesting that SNPs at these positions are genetically linked in all populations studied. Using functional studies with expression plasmids and recombinant IFN-β and IFNL3 proteins in liver-derived cell lines, the authors found evidence that the largest gene product, p179, appears to activate STAT signaling and induces the expression of ISGs.18 Furthermore, transient expression of p179 in hepatoma cells carrying an HCV replicon inhibited viral replication, indicating an antiviral role of p179. Taken together, these findings led the authors to conclude that the identified gene products may have a functional role in the innate immune response to RNA viruses. Furthermore, p179 shares 40.8% amino acid sequence similarity with IFNL3 and was designated as a new member of the interferon lambda family, called IFNL4.18
The results of this study have several implications: First, the study identifies a new starting point to better understand the role of IFNL in viral evasion. The correlation between the presence of a functional IFNL4 gene (i.e., ss469415590[δG]) and impaired clearance of HCV reported by Prokunina-Olsson et al. may suggest that IFNL4 is too weak to clear CHC. Furthermore, the authors conclude from this that weakly induced IFNL4 signals may reduce responsiveness of cells to IFN-β, thereby inhibiting efficient HCV clearance.18 Interestingly, the authors found that IFNL4 caused preactivation of interferon signaling which prevented further activation by IFN-β and IFNL3.18 This indicates that IFNL4 may cause refractoriness to IFN signaling, although it has been demonstrated that unlike IFN-β, hepatic IFNL signaling is resistant to refractoriness.17 IFNL4 appears to display different receptor binding sites compared to IFNL3 in regions required for the association with the second chain receptor of the IFNL receptor complex (IL10R2).18 This evidence led the authors to speculate that IFNL4 may engage a different receptor complex or act as a decoy cytokine competing with the classical IFNLs. The site of action of this possible cytokine might be expected to be the cell membrane surface, engaging its respective receptor complex to trigger JAK/STAT signaling. However, it seems that after induction by viral RNA mimics IFNL4 is merely enriched in the cytosol and it remains to be determined whether the protein is secreted to interact with cell surface receptors. Although intracellular overexpression of the IFNL4 protein appears to induce ISG expression, cells treated with recombinant protein failed to do so at significant levels. These findings indicate that further studies are needed to characterize the functional properties of the identified gene products, their mechanism of action, and their functional relevance in antiviral defense mechanisms.
Second, the study may provide insights into the understanding of the genetic evolution of innate immune responses. The authors found that the ss469415590[δG] frameshift variant encoding the putative IFNL4 is more common in AA patients and correlates with reduced HCV response in large clinical cohorts.18 The authors discussed the possibility that the beneficial allele ss469415590[TT] abrogating IFNL4 may have been selected during geographically distinct human evolution.18 It is tempting to speculate that the original purpose of the putative IFNL4 may have been important for the innate immune defense against other pathogens. It is conceivable that in an evolutionary context the “loss” of IFNL4 in humans occurred predominantly in geographic regions where the selective pressure from those pathogens eased. Moreover, since predominantly HCV-infected patients with genotype 1 were studied by the authors, a comprehensive multifactorial analysis of ss469415590 involving additional HCV genotypes, ethnic background, and geographic prevalence of different HCV genotypes may provide interesting insight into HCV evolution. For example, it will be interesting to determine whether more difficult-to-treat genotype 1 viruses co-evolved together with the selection of the ss469415590[TT].
Finally, the findings of the study may have clinical relevance for the management of patients with CHC. The recent groundbreaking discovery that SNPs in the region of IFNL3 are predictors for treatment outcome of CHC had revealed a new perspective for customized IFN-based therapies.8–16 In this context, potential algorithms have been proposed to utilize IFNL3 genotyping in the initial treatment decision making for CHC infections.19 Prokunina-Olsson et al. now extend these findings by revealing that ss469415590[δG] is more strongly associated with HCV clearance in AAs than the previously described SNPs, although it provides comparable information in patients with European and Asian ancestry.18 Including the upstream ss469415590 in the IFNL3 genotyping in the treatment decision algorithm may increase the reliability of prediction of IFN-based treatment outcome, especially in patients with African ancestry. Although IFNL3 genotyping appears to be of less relevance for predicting outcomes in IFN-free regimens, it seems to correlate with early viral kinetics.20 It will be interesting to assess the role of the novel SNP in IFN-free-based therapies. Further studies in additional cohorts with different genetic backgrounds and treatment protocols are needed to determine the role of ss469415590 in the management of HCV-infected patients and assess its clinical use in comparison with previously discovered biomarkers.
Beyond prediction of treatment response and customization of treatment strategies, another consequence of the newly discovered protein(s) could be their relevance as a potential drug target for clinical intervention in patients with the unfavorable ss469415590[δG] allele. Following a better understanding of the molecular mechanisms, it will be of interest to explore whether modulation of IFNL4 activity, e.g., by antagonizing IFNL4, may render patients with an unfavorable ss469415590 genotype more responsive to IFN-β and might thus enhance the efficacy of IFN-based therapies for HCV infection and other diseases including chronic HBV infection or cancer.
Taken together, the study by Prokunina-Olsson et al. provides a previously unknown starting point to understand the mechanisms of immune evasion during CHC and reveals new clues to understand the genetic evolution of innate immune responses in humans. Finally, the study may provide perspective for the development of improved biomarkers for the management of CHC. Despite promising IFN-β-sparing regimens being in clinical development, it is likely that a clinically relevant subset of difficult-to-treat patients may still require IFN-β in the future. Although the discovery of IFNL4 is likely very important, defining its detailed molecular mechanism will be key to integrate it into a broader context of IFN biology.
The authors acknowledge the support of Inserm, ANRS, the University of Strasbourg, the European Union (INTERREG-IV-Rhin Supèrieur-FEDER-Hepato-Regio-Net 2009 and 2012), DGOS, and the Laboratoire d'excellence HEPSYS (ANR-10-LAB-28).