Characterization of biological effect of IFN-$\lamda$4 in antiviral immune response

Abstract

IFN-mediated innate immune response represents the first line of defense against virus infection. There are three distinct interferon (IFN) families based on their structures and receptor complex. Type I IFNs include 13 IFN-$\alpha$s, IFN-$\beta$, IFN-$\omega$, IFN-$\varepsilon$, and IFN-$\kappa$ and they bind a heterodimeric transmembrane receptor composed of IFNAR1 and IFNAR2 subunits. IFN-$\gamma$ is a single member of type II IFN and its receptor is composed of IFNGR1 and IFNGR2. Type III IFNs, which were discovered in 2003, include IFN-$\lamda$1 (IL-29), IFN-$\lamda$2 (IL-28A), and IFN-$\lamda$3 (IL-28B). Recently, IFN-$\lamda$4 which is encoded on upstream region of IFNL3 on chromosome 19 was identified as a new member of type III IFNs. Type III IFNs exert their biological activity via a receptor complex that comprises the unique IFN$\lamda$R1 and IL-10R2 chain. Also, IFN-$\beta$ and IFN-$\lamda$s are known to be produced in virus infected cells.

In 2013, the IFNL4 gene which encodes IFN-$\lamda$4 was identified on upstream region of IFNL3 locus during GWAS with HCV infected patients. Interestingly, the production of functional IFN-$\lamda$4 is determined by germline dinucleotide frameshift variant in exon 1 of IFNL4 gene (rs368234815). While the rs368234815-$\delta$G allele produces the full-length IFN-$\lamda$4 protein by one of transcript variant, rs368234815-TT allele cannot make IFN-$\lamda$4 because of premature stop codon. In addition, this rs368234815-$\delta$G/TT allele is the strongest genetic marker to predict responsiveness to pegylated IFN-$\alpha$ based therapy. So current study is focused on the underlying mechanism of IFN-$\alpha$ unresponsiveness in HCV infected patients with rs368234815-$\delta$G allele.

Although Type I and III IFN binds to different receptor complex, both signaling results in activation of IFN-stimulated gene factor 3 (ISGF3) composed of phosphorylation of signal transducer and activator of transcription (STAT1), phosphorylated STAT2, and interferon regulatory factor 9 (IRF9). ISGF3 enters into nucleous and binds to IFN-stimulated responsive element (ISRE) in the promoter of the ISGs.

Using primary human hepatocytes and hepatoma cell lines, it was demonstrated that HCV infection induced IFN-$\lamda$4, which cause unresponsiveness to exogenous IFN-$\alpha$ treatment. It was reported that the gene expression of ISG15, one of the ISGs, was upregulated in HCV-infected liver and the expression level in the liver was higher in future nonresponder to pegylated-IFN-α based therapy than responder. Intracellular free ISG15 blocks SKP2 dependent degradation of USP18, which is an well-known negative regulator of type I IFN signaling, by preventing the ubiquitination of USP18 protein in human. Based on these previous reports, further study was done about the effect of IFN-$\lamda$4 on IFN-$\alpha$ responsiveness. It was proven that IFN-$\lamda$4 induced ISG15 stabilize the protein level of USP18, which is responsible for IFN-$\alpha$ unresponsiveness.

HCV infection is currently treated with DAA, which directly targets different viral protein. So, the effect of DAAs on the IFN-$\lamda$4 expression and responsiveness to IFN-$\alpha$ treatment was examined. DAA treatment to HCV infected PHH reduced the intracellular HCV RNA titer, causing dramatically attenuated the gene expression of IFN-$\lamda$4 as well as IFN-$\lamda$1. As the expression of IFN-$\lamda$s including IFN-$\lamda$4 is decreased by treatment of DAA, the unresponsiveness to IFN-$\alpha$ became restored. The data suggest that DAA treatment might be beneficial in combination with pegylated-IFN-$\alpha$ based therapy as rescue treatment of DAA-failed patients.

Unlike other IFNs, the secretion of IFN-$\lamda$4 is impaired, which in not due to a weak signal peptide. This property of poor secretion limits further investigation about IFN-$\lamda$4. Commercially available IFN-$\lamda$4 is purified from E.coli inclusion body through process including unfolding and refolding which might cause lack of proper protein folding. Also, recombinant IFN-$\lamda$4 protein without post translational modification, which is important process in mammalian cells cannot reflect biophysical properties of IFN-$\lamda$4.

In this study, to improve the secretion of IFN-$\lamda$4, de novo N-glycosylation sites were introduced to of IFN-$\lamda$4 sequence using the structure based glycoengineering approach. Fianlly IFN-$\lamda$4 can be purified from the culture supernatant by de novo N-glycosylation introduction. In addition, de novo N-glycosylation-introduced IFN-$\lamda$4 mutants provide stronger bioactivity including activation of JAK-STAT pathway and antiviral effect in viral infection model than commercially available IFN-$\lamda$4 from E.coli. Considering that activity of IFN-$\lamda$4 tends to be rapid, but transient compared to IFN-$\lamda$1, -$\lamda$2, and –$\lamda$3, de novo N-glycosylation-introduced IFN-$\lamda$4 mutants might be candidates for therapeutic or clinical application.