Interferons are members of the cytokine family which play a crucial role in the cellular defence against viral infection. Those of relevance in HCV infection include the type I IFNs (IFN-α subtypes and IFN-β), type II IFNs (IFN-γ) and type III IFNs (IFN-λ, otherwise known as interleukin (IL)-29, IL-28A and IL-28B). IFN-α and IFN-β induce the innate immune response, and can directly inhibit viral replication. Additionally, they are able to direct the adaptive immune response by priming cytotoxic T cells and T helper cells. IFN-γ has immunomodulatory activity and is secreted by NK cells and cytotoxic T cells (Lemon S, 2010). HCV RNA replication induces type I IFN within 48 hours of infection in a chimpanzee model (Bigger et al., 2001). It is postulated that this early IFN response may restrict viral replication in acute infection (Neumann-Haefelin et al., 2005). However, despite this early response, type I IFN does not play a major role in viral clearance and HCV is largely eliminated by non-cytolytic effects through the IFN-γ driven interferon-stimulated gene (ISG) expression in hepatocytes. IFN-γ may also inhibit HCV infection by downregulating claudin-1 and CD81 (Wei et al., 2009).
The host innate immune response is induced upon recognition of conserved molecular patterns expressed on microbial and viral structures (pathogen associated molecular patterns; PAMPs) by resident membrane and cytosolic receptor molecules (Olivieri et al., 2013a). Amongst these molecules are the toll-like receptors (TLRs) which are transmembrane proteins of mammalian cells. In humans, viral nucleic acids are recognised by the endosomal TLRs -3, -7, -8 and -9, by PKR, and by the cytosolic retinoic acid-inducible gene-like receptors
(RLRs) retinoic acid-inducible gene I (RIG-I) and melanoma differentiation- associated gene (Mda5).
HCV RNA contains specific PAMPs, including stem-loop double-stranded RNA (dsRNA) and poly-uridine motifs in the 3’-UTR (Tuplin et al., 2002). Cell culture studies have suggested that two distinct PAMP receptors, TLR-3 and RIG-I, direct the host response to HCV infection (Ishii and Koziel, 2008, Lemon, 2010). TLR-3 acts through the TIR domain containing adaptor inducing interferon-β (TRIF) molecule, whereas TLR-7, -8 and -9 act through myeloid differentiation primary- response gene 88 (MyD88) (Olivieri et al., 2013a). The association of TLR-3 with TRIF leads to the recruitment of TNF receptor-associated factor (TRAF) 6, TRAF3 and the nuclear factor-kappa B (NF-κB) activator binding kinase 1 (TBK1),
resulting in phosphorylation of interferon regulatory factor 3 (IRF-3) by TBK1 and inhibitor of kappa B kinase-related kinase (IKKε). RIG-I is a DEAD box helicase able to sense short HCV replication intermediate dsRNAs with free 5’-
triphosphatases. On recognition of the HCV PAMP, it is recruited to the mitochondrial surface where it interacts with MAVS (mitochondrial antiviral signalling protein, also known as IPS-1, VISA and Cardif) through shared caspase recruitment domains (CARDs) resulting in recruitment of TBK1 and IKKε. Both the E3 ubiquitin ligase triple motif-containing protein (TRIM) 25 and the
mitochondrial targeting chaperone protein 14-3-3ε are involved in this process; TRIM25 mediates the ubiquitination of RIG-I at position Lys-63 which facilitates MAVS binding and is important for IFN production (Loo et al., 2006). RIG-I also interacts with apoptosis-associated speck like protein containing CARD resulting in activation of a caspase-1-dependent inflammasome (Nitta et al., 2013) (figure 1-6).
Cellular recognition of the HCV PAMPs by various PAMP receptor pathways leads to the activation and nuclear translocation of NF-κB and IRF-3. IRFs control the induction of IFN at the transcriptional level by binding to specific promotor elements. Although IRF-5 and IRF-7 are also important in the host response to viral infection, their role in HCV infection has not been characterised. Together with NF-κB and ATF2/c-Jun, IRF-3 forms an enhanceosome complex on the IFN- α/β promoter, resulting in IFN-β expression and secretion from the infected cell. IRF-3 also controls the transcription of the IFN-λs.
Both IFN-α and IFN-β are recognised by the type I IFN-α/β receptor (IFNAR) resulting in activation of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. Following IFNAR binding, STAT1 and STAT2 are phosphorylated to form a heterodimer, resulting in recruitment of IRF9 and formation of the ISGF3 transcription factor complex. ISGF3 binds to the interferon-sensitive response element (ISRE) and results in high level of
expression of ISGs (Ishii and Koziel, 2008, Lemon, 2010). Upregulation of ISGs has been demonstrated in the liver biopsy specimens of patients with CHC (Sarasin- Filipowicz et al., 2008), and is associated with an increase in circulating
cytokines, including chemokine IFN-γ inducible protein-10 (CXCL10/IP-10)
(Askarieh et al., 2010). Interestingly, Robinson and colleagues (2015) found that Gt1-infected biopsies demonstrated an upregulation of genes regulated by type I and III IFNs, whereas Gt3-infected biopsies displayed upregulation of IFN-γ
induced transcripts.
Figure 1-6: Induction of innate immunity by HCV in hepatocytes. Extracted from Wong et al. (2016). IFNAR, type 1 IFN-α/Β receptor; TLR3; toll-like receptor 3; P, phosphorylated; ISGF3, interferon-stimulated gene factor 3.
The gene products of ISGs are able to limit HCV replication at different phases of the virus life cycle, as well as having direct effects on cellular processes. Many PAMP receptors and related molecules are also ISGs, for instance, RIG-I and PKR. Interferon-induced transmembrane protein 1 (IFITM1) is an important ISG
effector, which is induced during IFN-α treatment and accumulates at hepatic tight junctions where it interacts with CD8 and OCLN to disrupt HCV entry (Wilkins et al., 2013).
ISG15 is another important ISG, which modulates host antiviral responses through ‘ISGylation’ of viral and host proteins and inhibition of virus release. IRF3, RIG-I and PKR are all modified by ISGylation, resulting in positive
regulation of type I IFN signalling. ISG15 also targets many proteins involved in the innate immune response to viral infection, including JAK1, STAT1 and other ISGs (Morales and Lenschow, 2013). The stability of HCV NS5A protein is
decreased by IFN-mediated ISGylation, leading researchers to suggest that ISG15 could be used as a therapeutic adjunct to IFN-α treatment in CHC (Kim and Yoo, 2010). Additionally, knockdown of ISG15 expression is associated with increased HCV replication in various cell lines (Jones et al., 2010, Domingues et al., 2015). Conversely, it has also been proposed that increased ISG15/ISGylation may promote HCV infection (Chen et al., 2010). Early PKR-dependent induction of ISG15 may inhibit RIG-I ubiquitination, blocking subsequent HCV RNA-mediated RIG-I activation and leading to negative regulation of the host immune response and promotion of HCV replication (Arnaud et al., 2011).
HCV utilises a number of additional strategies to evade host immune
surveillance. Core protein is able to downregulate IFN signalling by blocking STAT1 phosphorylation, preventing heterodimerisation with STAT2 (Lin et al., 2006). Foy et al. (2003) demonstrated that the NS3/4A protein complex is able to block IRF-3 activity in vitro through preventing accumulation of the
phosphorylated IRF-3 isoform, and the nuclear translocation of IRF-3, thus promoting viral persistence. Additionally, NS3/4A is able to cleave host proteins including TRIF and the adaptor protein MAVS, releasing it from the mitochondrial membrane and blocking its ability to signal (Lemon, 2010, Bowie and
Unterholzner, 2008). Recent data suggest that immune evasion may also be mediated through NS3-4A targeting of importin β1 and related modulators of IRF3 and NF-κB nuclear transport (Gagne et al., 2017). Abe et al. (2007) showed
that the NS5A protein was able to inhibit TLR-MyD88-induced signalling in mouse macrophage cells by binding to the death domain of MyD88. Overexpression studies have suggested that the viral protein NS4B may also interfere with RIG-I signalling (Tasaka et al., 2007).
A key component of the IFN antiviral response is PKR. PKR phosphorylates serine 51 of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2α) leading to blockage of protein synthesis initiation and inhibition of viral replication (Gale et al., 1998). PKR is also a mediator of stress-induced apoptosis and repression of PKR leads to malignant transformation of mammalian cells (Koromilas et al., 1992). The viral protein NS5A is able to bind to and inhibit PKR, blocking
apoptosis through sequestration of p53, modulation of intracellular electrolyte levels, binding to growth factor receptor-bound protein 2 and induction of IL-8. The E2 protein also appears to bind to and inhibit PKR through molecular
mimicry, as a result of sequence similarity to the phosphorylation domains of PKE and eIF-2α (Taylor et al., 1999).
NK and NKT cells are an important component of the innate immune response and contribute to suppression of viral replication through cytolysis of infected cells, production of immunomodulatory cytokines, such as IFN-γ, and activation of dendritic and T cells (Ishii and Koziel, 2008). It has been shown that certain NK cell receptor genes (killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3) and human leukocyte antigen (HLA)-C1) are associated with resolution of HCV infection, suggesting that inhibitory NK cell interactions are important in determining outcome of infection (Khakoo et al., 2004). In CHC, NK cells demonstrate alterations in phenotype and function (Rehermann, 2013), with higher levels of STAT1, increased degranulation and decreased production of IFN-γ and TNF-α compared to NK cells from healthy controls. DAA therapy is associated with a rapid normalisation of NK cell cytotoxic effector functions, a decrease in the expression level of the ISG STAT1, and a decrease in serum levels of CXCL10/IP-10 and CXCL11 (Serti et al., 2015).
Several genetic polymorphisms have been shown to influence the outcome of HCV infection, namely those encoding IFN-λ, IFN-γ, and the NK receptor KIR2DL3 and its HLA ligand as described above (Lemon, 2010). Genome wide association studies (GWAS) found a strong correlation between single nucleotide
polymorphisms (SNPs) near the IL-28B (recently renamed the IFN-λ3 or IFNL3) gene (rs12979860 and rs8099917) and rates of spontaneous and IFN-based treatment-associated clearance of HCV (Ge et al., 2009, Rauch et al., 2010, Suppiah et al., 2009). HCV Gt1-infected patients with the unfavourable IFNL3 rs12979860 TT genetic polymorphism had significantly lower sustained virologic response (SVR) rates in response to IFN-based treatment than patients with the CC genotype. It has previously been demonstrated that patients with IFNL3 non- responder genotypes (non-CC) have higher hepatic ISG expression at baseline than those with responder genotypes (Urban et al., 2010), and that this
correlates with a poorer response to IFN-α based therapy (Asselah et al., 2008). Furthermore, analysis of transcription factors in the peripheral blood of patients with CHC suggests that those with IFNL3 responder genotypes have a Th1/NK dominant state favouring viral clearance (O'Connor et al., 2016).
In 2013, a new polymorphism rs368234815 (previously ss469415590; TT or ΔG) upstream from the IFNL3 gene on chromosome 19q13 was found to result in a frameshift mutation, creating the ORF for a novel inducible protein-coding gene, IFNL4, and resulting in transient expression of the IFN analogue, IFN-λ4
(Prokunina-Olsson et al., 2013). The rs368234815-ΔG allele was found to
correlate with the previously identified rs12979860-T allele, and more strongly predicts HCV clearance in Gt1 infection (O'Brien et al., 2015). These SNPs can result in the production of no IFN-λ4 protein (TT genotype), a mutant IFN-λ4 protein with reduced antiviral activity (called P70S), or the wild type IFN-λ4 protein. Counterintuitively, individuals with the rs368234815-ΔG allele who produce the active wild type IFN-λ4 protein have significantly lower rates of spontaneous viral clearance. IFN-λ4 induces STAT1 and STAT2 phosphorylation, and activates ISGs. This pre-activation of IFN-signalling may prevent further activation in response to viral infection, and may impair the response to exogenous IFN-α as described above (Prokunina-Olsson et al., 2013). In Gt1- infected biopsies, the presence of the IFNL4 ΔG variant was associated with higher expression of the ISGs IFIT1, ISG15, RSAD2 and USP18. However, IFNL4 genotype did not influence baseline ISG expression in Gt3-infected biopsies, perhaps explaining the reduced predictive power of IFNL3 genotype in Gt3 infection (Mansoor et al., 2016, Cariani et al., 2016).