The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs.
ABSTRACT: IFNL3, which encodes interferon-?3 (IFN-?3), 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.
Project description:BACKGROUND & AIMS:Genetic polymorphisms within the interferon lambda (IFN-?) region are strongly associated with hepatitis C virus (HCV) clearance; the IFNL4-?G/TT (rs368234815) polymorphism, which controls the generation of IFN-?4 protein, is more strongly associated with HCV clearance than rs12979860 (the 'IL28B variant'). An IFNL3 3' untranslated region polymorphism (rs4803217) has been proposed as a causal variant that may affect HCV clearance by altering IFNL3 mRNA stability. METHODS:We compared IFNL4-?G/TT and rs4803217 for association with response to pegylated-IFN-?/ribavirin in the VIRAHEP-C and HALT-C trials, and spontaneous HCV clearance in the ALIVE, UHS and WIHS studies. Genotyping was performed with TaqMan assays. We compared differences in mean reduction in HCV RNA levels by genotype and haplotype. For HCV clearance, we calculated p-values comparing c-statistics for IFNL4-?G/TT and rs4803217 genotypes by a bootstrap approach. RESULTS:Among European Americans, linkage disequilibrium between IFNL4-?G/TT and rs4803217 was strong (r(2)=0.89-0.99) and there were no significant differences between the variants. In African American (AA) individuals enrolled in VIRAHEP-C, HCV RNA at treatment day 28 was more strongly associated with IFNL4-?G/TT than rs4803217 (p=0.003); the IFNL4-?G:rs4803217-G haplotype, which includes the putatively favorable IFNL3 allele, was actually associated with the poorest day 28 response (p=0.03, comparison to IFNL4-?G:rs4803217-T haplotype). Among AA participants, associations were stronger for IFNL4-?G/TT than rs4803217 for undetectable HCV RNA at week 24 in Virahep-C (p=0.03) and week 20 in HALT-C (p=0.03), as well as for spontaneous HCV clearance (p=0.048). CONCLUSION:IFNL4-?G/TT is the primary IFN-? region polymorphism for impaired HCV clearance.
Project description:Genetic polymorphisms in the region of the interferon-λ genes (IFNL) associate with clearance of hepatitis C virus (HCV) infection. One of these polymorphisms, IFNL4 rs368234815, determines loss or gain of function of the IFNL4 gene by frameshift variation. The very same and a second one, IFNL3 rs4803217, are supposed to impact the expression of IFNL3: while IFNL4 rs368234815 is suggested to modulate IFNL3 transcription, IFNL3 rs4803217 is thought to alter IFNL3 mRNA stability. The latter process is believed to be partially driven by an HCV-induced ectopic expression of myosin heavy chain genes 7B and 7 and their co-expressed microRNAs mir499 and mir208B. These ideas are evidenced by functional investigations on peripheral blood mononuclear and hepatoma cells in culture. Our study aimed at exploring IFNL3 gene expression in clinical samples, i.e., in ex vivo derived liver tissue from patients with chronic hepatitis C (n = 57) and various other diseases (n = 56). By applying an assay designed to specifically quantify IFNL3 and discriminating paralogous IFNL2 transcripts, IFNL3 mRNA expression was not found to differ significantly between chronic hepatitis C and control samples. Among patients with chronic HCV infection, moreover, IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles did not associate with reduced IFNL3 gene expression. Finally, myosin heavy chain genes 7B and 7 and corresponding microRNAs mir499 and mir208B were not found activated in liver in chronic HCV infection. Of note, detectability of MYH7 mRNA related to the procedure of liver biopsy sampling, as tissue obtained by direct punctation of the liver during laparoscopic inspection was less likely to contain MYH7 transcripts than samples acquired by percutaneous punctation. In conclusion, data on ex vivo derived liver tissue samples argue against an attenuating impact of IFNL3 rs4803217 or IFNL4 rs368234815 minor alleles on hepatic IFNL3 gene expression in vivo.
Project description:BACKGROUND:Variants of the interferon-lambda3 (IFNL3) gene have been associated with both spontaneous and treatment induced clearance of HCV infection. Attempts to link polymorphisms of the IFNL3 gene with variation in the level of IFNL3 expression have been inconclusive. This is partially due to the difficulty to design assays distinguishing IFNL3 from IFNL2. METHODS:In this study an allele specific real-time PCR (RT-PCR) assay was developed which allows the relative quantification of the two IFNL3 transcripts in cells heterozygous for SNP IFNL3.rs4803217 in the 3'UTR of the IFNL3 gene. This SNP is in strong linkage disequilibrium (LD) with the predictive marker rs12979860. RESULTS:Raji cells showed two-fold increased levels of IFNL3.rs4803217 C-allele expression. In peripheral blood mononuclear cells (PBMCs) of eight uninfected donors, two donors showed increased IFNL3.rs4803217 C-allele expression. CONCLUSION:This indicates that allele specific differences in IFNL3 expression vary between individuals and might contribute to the variety of outcomes in HCV infected patients.
Project description:Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3' untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3' UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217?G allele mRNA forms well-defined 3' UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure.
Project description:AIM:To evaluate the association of IFNL3 (IL28B) SNP rs4803217 with severity of disease and treatment outcome in chronic hepatitis C (CHC). METHODS:The study enrolled 196 CHC Polish patients (82 women and 114 men in age 20-64) infected with hepatitis C virus (HCV) genotype 1. They were treatment naïve and qualified to pegylated interferon alpha (PEG-IFN-?) and ribavirin (RBV) therapy. The analyzed baseline parameters included: degree of inflammation, stage of fibrosis, viral load as well as alanine aminotransferase (ALT), asparagine aminotransferase (AST) and total bilirubin (TBIL). The analysis of response to therapy included: sustained virological response (SVR), defined as undetectable serum HCV RNA level six month after completion of 48-wk therapy, and relapse, defined as achieving undetectable viral load at the end of treatment but not SVR. HCV genotyping and HCV RNA quantification were performed using commercially available tests. DNA was isolated from peripheral blood mononuclear cells or from buccal cell swabs. In addition to rs4803217, also single nucleotide polymorphisms (SNPs) (rs12979860, rs8099917 and rs12980275) of known significance in predicting of HCV clearance were analyzed. SNPs were determined by high resolution melt analysis and confirmed by sequencing of amplicons. RESULTS:Frequency of rs4803217 genotypes in studied group was as follows: 27.55%; 54.59% and 17.86% for CC, CA and AA, respectively. The rs4803217 SNP, similar to other analyzed SNPs, was not associated with severity of CHC (grade of inflammation, stage of fibrosis, baseline viral load as well as biochemical parameters: ALT, AST, TBIL). It was demonstrated that the rs4803217C allele is associated with SVR (C vs A: P < 0.0001; dose of C allele: P = 0.0002) and non-relapse (C vs A: P = 0.001; dose of C allele: P = 0.002). Moreover, it was found that patients with CC genotype have significantly higher response rates as compared with CA/AA patients (P < 0.0001), whereas patients carrying A allele are significantly predisposed to relapse after treatment (P = 0.0007). Moreover, the association of rs4803217 with SVR was comparable to that of rs12979860 and stronger as observed for rs12980275 and rs8099917. Association of rs4803217 with relapse, was the strongest as compared with the other SNPs. The analysis of combined rs4803217 and rs8099917 genotypes demonstrated that additional genotyping of rs8099917 had no significant impact on the prediction of SVR. Multivariate analysis revealed that among analyzed SNPs only rs4803217 is an independent predictor of SVR (P = 0.016) and relapse (P = 0.024). CONCLUSION:The rs4803217 SNP is a strong, independent and superior predictor of SVR and relapse in HCV genotype 1 infected CHC patients treated with PEG-IFN-? and RBV.
Project description:Background:The association between interferon lambda-3 (IFNL3?also known as interleukin 28B, IL28B) rs12979860 polymorphism and the development of hepatocellular carcinoma (HCC) has been investigated in recent studies with inconclusive and inconsistent results. IFNL3 rs12979860 polymorphism has been shown a marked differential distribution with regional and ethnic variation. Whether this single nucleotide polymorphism influences susceptibility to hepatitis C virus (HCV)-related HCC remains elusive. Methods:In this case-control study, a total of 157 Chinese Han patients with chronic HCV infection were enrolled, including 62 HCV-related HCC patients and 95 chronic hepatitis C (CHC) patients without HCC, and the genetic polymorphism of IFNL3 rs12979860 was genotyped via a DNA microarray-based assay. The logistic regression analysis was employed to determine the correlation between the genetic polymorphism and risk of HCV-related HCC. Results:A higher proportion of CT/TT genotype and T allele was observed in HCC patients compared to the CHC group. Under the genetic model of allele frequency, the T allele was associated with elevated risk of HCV-related HCC in the Chinese population compared to C allele after an adjustment for age, gender, body mass index, HCV infection duration, and HCV genotypes (P=0.046). In the subgroup analysis stratified by HCV genotype, subjects with CHC genotype 1b infection carrying rs12979860 T allele and CT+TT genotype had higher susceptibility to HCC than those with C allele and CC genotype (P=0.020, P=0.037, respectively). Conclusion:IFNL3 rs12979860 polymorphism with T allele could be a factor that increases the risk of HCV-related HCC in the Chinese population, especially those subjects with CHC genotype 1b infection.
Project description:<h4>Background</h4>In non-uremic subjects, IFNL4 rs368234815 predicts HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in dialysis subjects. We also evaluated an association of rs368234815 with patients` survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms.<h4>Methods</h4>Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ?G/TT, ?G/?G) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. We used the Kaplan-Meier method with the log-rank test for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software.<h4>Results</h4>The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ?G allele carriers (2.63, 1.38-5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45-5.43, 0.002 for rs12980275 and 2.44, 1.27-4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69-24.2, 0.003). Survival analysis suggested an association of the ?G allele with mortality due to neoplasms (log-rank P?=?0.005). The rs368234815 ?G allele caused TFBS removal for PLAGL1.<h4>Conclusions</h4>In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, identifying the homozygosity in the variant ?G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that we observe in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harbouring the rs368234815 ?G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.
Project description:UNLABELLED:The microRNA miR-122 is highly expressed in the liver and stimulates hepatitis C virus (HCV) replication in vitro. IFNL3 (lambda-3 interferon gene) polymorphisms and the expression of miR-122 have been associated with sustained virological response (SVR) to treatment with pegylated interferon plus ribavirin in patients with chronic hepatitis C (CHC). We investigated, in vivo, the relationship between miR-122 expression, IFNL3 polymorphism, fibrosis, and response to PEG-IFN plus ribavirin. Pretreatment liver biopsy specimens and serum samples from 133 patients with CHC were included. Sixty-six patients achieved SVR, and 64 failed to respond to the treatment (43 nonresponders [NR] and 21 relapsers [RR]). All stages of fibrosis were represented, with 39, 50, 23, and 19 patients, respectively, having Metavir scores of F1, F2, F3, and F4. miR-122 expression was assessed by real-time quantitative PCR (RT-qPCR) and IFNL3 rs12979860 by direct sequencing. Hepatic miR-122 expression was higher in patients with the IFNL3 CC genotype than in those with the IFNL3 CT or TT genotype, in all patients (P = 0.025), and in NRs plus RRs (P = 0.013). Increased hepatic miR-122 was more strongly associated with complete early virological response (cEVR) (P = 0.003) than with SVR (P = 0.016). In multivariate analysis, increased hepatic miR-122 was only associated with the IFNL3 CC genotype. miR-122 was decreased in patients with advanced fibrosis (Metavir scores of F3 and F4) compared to its levels in patients with mild and moderate fibrosis (F1 and F2) (P = 0.01). Serum and hepatic expression of miR-122 were not associated. The association between miR-122 and IFNL3 was stronger than the association between miR-122 and response to treatment. miR-122 may play a role in the early viral decline that is dependent on IFNL3 and the innate immune response. IMPORTANCE:miR-122 plays a crucial role during HCV infection. Moreover, it was reported that miR-122 binding within the HCV genome stimulates its replication. Moreover, miR-122 is highly expressed within hepatocytes, where it regulates many cellular pathways. A reduction of miR-122 expression has been suggested to be associated with responsiveness to IFN-based therapy in patients with chronic hepatitis C. Several independent genome-wide association studies reported a strong association between IFNL3 polymorphism and responsiveness to IFN-based therapy. We report here a strong association between the expression of miR-122 and IFNL3 polymorphism that is independent of the response to the treatment. Our data suggest that modification of miR-122 expression may play an important role in the molecular mechanism associated with IFNL3 polymorphism. Moreover, we report a reduction of miR-122 at more advanced stages of fibrosis in patients with chronic hepatitis C.
Project description:BACKGROUND:Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive. METHODS:IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3. RESULTS:PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07). CONCLUSIONS:Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection.
Project description:The purpose of the present review is to summarise the current knowledge on the association between single nucleotide polymorphisms (SNPs) in the interferon L3 (IFNL3) gene and hepatitis C virus (HCV) infection in children. Many studies in adults have demonstrated that genetic variation in IFNL3 is a strong predictor of the virological response in treatment-naive patients with HCV genotype 1 who were treated with Pegylated-IFN-? and ribavirin. Genetic variation in IFNL3 is also associated with the spontaneous clearance of HCV. Thus far, few paediatric studies have explored the association between variations in the IFNL3 gene and either spontaneous or treatment-induced clearance of HCV. The CC genotype of the rs12979860 SNP is associated with the spontaneous clearance of HCV in children independently of HCV genotype. Four paediatric studies have shown that both the CC genotype of the rs12979860 SNP and the TT genotype of the rs8099917 SNP are associated with the treatment-induced (IFN monotherapy and Pegylated-IFN-? and ribavirin association) clearance of HCV, while the rs12980275 SNP did not affect the virological response. The possible role of IFNL3 gene variation as a pre-treatment and on-treatment predictor of virological response in children is highly attractive but still undetermined. Further paediatric studies are needed to evaluate if testing for SNPs in IFNL3, either alone or together with other predictors of response to treatment, could be used to direct treatment strategies, including an avoidance of unnecessary protease inhibitor therapy and the duration of treatment.