ISGs are genes whose transcriptional output increases in response to virus-induced IFN, largely due to the presence of ISRE sequences in promoter and enhancer regions. Their function is to establish a cellular anti-viral state to efficiently limit further replication and spread of the virus (Randall and Goodbourn, 2008). ISGs can represent up to 5% of the total number of cellular genes, however, this percentage may be underestimated as it only represents genes with changes in mRNA abundance. Since several ISGs are present in
multiple isoforms and give rise to proteins with differences in cellular localization and activity, some ISGs changes upon type I IFN stimulation may remain undetected.
ISGs take on a wide range of activities. Many will control viral infection by directly targeting pathways and functions required during pathogen life cycles. Indeed, to complete their life cycle, viruses must enter cells, replicate their genome, and exit in order to infect new cells. Every stage of the virus life cycle is a potential target for ISGs intervention. In addition, a number of ISGs also act to enhance pathogen detection and innate immune signalling, while others encode for pro-apoptotic proteins leading to cell death under specific conditions. Of these ISGs, several have been identified as having direct anti-IAV activities.
Among important and well-studied proteins encoded by ISGs, the myxovirus resistance proteins (Mx1 and Mx2) are considered important in the defence against IAV infection. Furthermore, diversity of equine Mx gene has been associated with variations in susceptibility vis-a-vis resistance against EIV evidence, which suggests an important role of these proteins for the resistance to influenza virus infection (Manuja et al., 2014). Mx1 and Mx2 belong to a small family of dynamin-like large guanosine triphosphates (GTPases). The former broadly inhibits IAV and acts prior to genome replication at a post-entry step in the virus life cycle. It contains a middle stalk domain and a GTPase effector domain, which are both essential for self-oligomerization and formation of ring-like structures that trap incoming viral components, such as NP. In doing so, Mx1 inhibits nuclear import of vRNP, and potentially directs them to sites of degradation. Mx2 has been characterized more recently as an antiretroviral effector protein against HIV but has less potent or no antiviral activity against IAV (Busnadiego et al., 2014).
Many of the proteins involved in ISGylation and de-ISGylation are also induced by type I IFN (i.e. TRIM25, ISG25, USP18, …) (Sgorbissa and Brancolini, 2012). ISGylation can positively or negatively affect the targeted protein. In the case of IRF3 it increases stability by preventing poly-ubiquitination and leads to sustained transcription factor activity. In contrast, ISGylation of cyclin-dependent kinase 1 (CDK1) leads to protein destabilization and cell cycle inhibition. It is believed that ISGylation is a general, non-specific mechanism of host defence, which potentially impacts all viral proteins translated in IFN-stimulated cells (Durfee and Huibregtse, 2010). A small IFN-induced ubiquitin-like protein, Interferon- stimulated gene 15 (ISG15) is one of the most highly induced ISGs. However, despite reports describing ISG15 function and targets in vitro, its role in vivo remains controversial
(Bogunovic et al., 2012). ISG15 is also secreted from immune cells and enhances the production of type I IFN (Bogunovic et al., 2013). Removal of ISG15 conjugates (deISGylation) is performed by USP18. USP18 maintains long-term IFN desensitization through an interaction with IFNAR-II, which suggests that USP18-mediated inhibition may be restricted to type I IFN signalling (Schneider et al., 2014).
The tripartite motif (TRIM) family of proteins are also induced by virus-induced IFNs. This family is composed of more than 60 members and exhibits a wide range of activities including E3 ubiquitin ligase activity, SUMOylation and ISGylation. Among them is found TRIM25 responsible for ubiquitination and activation of RIG-I as described previously (section 1.2.1).
Another important ISG-encoded protein is the 2’-5’-oligoadenylate synthase (OAS). Upon binding dsRNA, OAS becomes enzymatically active and converts ATP into 2’-5’- oligoadenylate, which then functions as a second messenger to activate latent RNase L. OAS and activated RNAse L act together to degrade viral RNA in the cytosol. They also generate short fragments with 3’ monophosphates, which lead to protein synthesis inhibition and viral growth arrest. By-products of this degradation might also be recognized by RLRs, hence participating in the positive feedback in the IFN response (Silverman et al., 2007). In accordance with this, the magnitude of IFN induction is enhanced by the activation of RNase L (Malathi et al., 2007).
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an RNA-binding protein kinase constitutively expressed in an inactive conformation in mammalian cells. The binding of dsRNA to PKR release its auto-inhibition and thereby activates it. PKR also becomes upregulated upon interferon treatment (Hovanessian, 1989). Once activated, it phosphorylates a number of substrates and activates different signal transduction pathways to counteract potential threats. PKR notably phosphorylates the a–subunit of eukaryotic translation initiation factor 2a (EIF2a) in parallel of binding viral dsRNA, thus inhibiting translation of both cellular and viral proteins (Garcia et al., 2006). Moreover, PKR plays an important role in signal transduction and transcriptional control through activation of IkB, the inhibitor of NF-kB. Activation of PKR in infected cells results in apoptosis, cell growth arrest and autophagy, all of which limit viral replication and spread in the host. In addition, PKR has been shown to stabilize IFN-a/b mRNAs, thereby ensuring robust type I IFN production (Munir and Berg, 2013).
The Cholesterol 25-Hydroxylase (CH25HC) gene is also upregulated by type I IFNs. The product of this gene is an enzyme that converts cholesterol into 25-hydroxycholesterol (25HC). CH25H mediates protection against IAV at an early stage in the infectious cycle, possibly at the step of virus-host membrane fusion (Schoggins and Randall, 2013). However, 25HC may impact virus infection by additional mechanisms, such as directly altering membrane properties, inhibiting sterol biosynthesis, and affecting prenylation of both virus and host proteins, which plays an important role in the virus life cycle (Matsumiya et al., 2013). In addition, high concentrations of 25HC change the physical properties of membranes, which prevents virus-host membrane fusion (Matsumiya et al., 2013).
The only ISGs shown to have a bona fide role in blocking virus entry are members of the IFN-inducible transmembrane (IFITM) family. The IFITM family of proteins is composed of four members, IFITM1, IFITM2, IFITM3 and IFITM5, that have been shown to be potent inhibitors of IAV infection (Brass et al., 2009). These proteins are enriched in late endosomes and lysosomes. The most potent member of the IFITM family against IAVs is IFITM3, which interfere with fusion between viral and endosomal membranes thereby limiting the viral entry (Brass et al., 2009, Everitt et al, 2012).
During late stages of the virus life cycle, viral nucleic acids are packaged into capsids, and viral particles exit cells either by cell lysis, exocytosis, or direct budding from the plasma membrane. Relative to other stages in the virus life cycle, few ISGs are known to inhibit viral assembly and viral egress. Viperin, encoded by the ISG RSAD2, is one of the best-studied, most highly induced antiviral effectors. It can be induced by at least two different innate immune pathways, such as the JAK-STAT signalling pathway or via direct activation by IRF3. Viperin normally resides in the endoplasmic reticulum (ER) and in ER- derived lipid droplets, organelles important for lipid metabolism. Viperin inhibits IAV budding at the host cell membrane by inhibiting farnesyl diphosphate synthase (FFPS), an enzyme involved in isoprenoid biosynthesis (Wang et al., 2007).
Another ISG-encoded protein important at the late stage of the virus life cycle is Tetherin. This protein is encoded by the ISG BST2, and it inhibits virus budding by using two membrane anchors to trap virions on the plasma membrane (Swiecki et al., 2011).
Finally, PRRs, IRFs, and several other signal transducing proteins, such as JAK, STAT1/2 and IRF9, are present at baseline, but are also ISGs and reinforce the IFN response. Upon induction, this set of ISGs are directly induced by IRF activation independently of the
JAK-STAT pathway, which likely evolved to counteract pathogen strategies of immune evasion (Iwasaki and Pillai, 2014).