In the healthy respiratory tract, the epithelium is ciliated and covered by a protective layer of mucus, which limits attachment of viral particles to the epithelium and entraps and transports them upward through the action of ciliary apparatus to the pharynx where they are swallowed. Once swallowed, viral particles are destroyed in the stomach by
cytoplasm nucleus 1. Virus entry 2. Endosomal release 3. Nuclear import (-) vRNP (+) cRNP (+) mRNA Cap An 7. vRNA export 4. mRNA export (+) mRNA Cap An 8. vRNA transport 9. Virion assembly 5. Viral protein translation
6. Viral protein import 10. Viral budding HA NA PA PB2 PB1 NP M2 M1 NS1 NEP endosome
the action of acidic secretions and digestive enzymes (Myers C, 2006). A normal architecture of the epithelium is essential for successful exclusion of viruses. However, if EIV breaks through this first line of defence and infects host cells, it will strongly compromise this epithelial barrier. Danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) will in turn be recognised by pathogen recognition receptors (PRRs) and alert the immune system that a viral invasion is occurring.
1.2.1. Detection of IAV
Influenza virus is recognized by at least 3 distinct classes of PRRs: Toll-like receptors (TLR), NOD-like receptor family member NOD-LRR- and pyrin domain-containing 3 (NLRP3), and cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs).
The Toll-like receptor family of transmembrane proteins are able to recognize a wide range of microbial ligands, and signal downstream through either myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-b (TRIFs) to stimulate expression of anti-viral cytokines, such as Interferons (IFNs) (see section 1.2.3 for more details about IFNs). Innate responses to IAV infection are primarily triggered by recognition of viral nucleic acids by endosomal TLR3 and TLR7. Their respective location enables the detection of viral nucleic acids present in the extracellular environment or produced by uncoating or degradation of viral particles (Wu et al., 2011). These features allow to mount an anti-viral response without the need for viral replication.
TLR3 has a relatively wide tissue distribution and is highly expressed in myeloid dendritic cells (mDCs) (Alexopoulou et al., 2001). Engagement of TLR3 by IAV dsRNA triggers a complex signal-transduction pathway starting with the dimerization and tyrosine phosphorylation of TLR3 (Sarkar et al., 2004), followed by the recruitment of TRIF (Yamamoto et al., 2003, Hoebe et al., 2003) and phosphatidylinositol 3 kinase (PI3K) (Sarkar et al., 2004). Engagement of TRIF activates both NF-kB and IRF-3 transcription factors, which in turn leads to IFN induction (Hardy et al., 2004).
TLR7 is expressed in few cell types and exclusively in endosomes (Kaminski et al., 2012). Ligands for this TLR include single-stranded RNA molecules (ssRNA) with no sequence specificity (Lund et al., 2004), but requiring the presence of several uridines in close proximity (Diebold et al., 2006). The mechanism of IFN induction follows a different profile from dsRNA-induced activation of TLR3, in that ssRNA-activated TLR7 recruits the MyD88 adaptor, which in turn recruits a complex containing the kinases interleukin-1 receptor- associated kinase 4 (IRAK-4), IRAK-1 and TRAF6 (Hacker et al., 2006), and leads to activation of NF-kB and induction of IFN. During an IAV infection TLR7 is particularly important in
plasmacytoid dendritic cells (pDCs), while it is dispensable in other cell types (Yoneyama et al., 2004, Jeisy-Scott et al., 2012), either because it is not expressed (Mayer et al., 2007) or because its localization is non-endosomal (Ioannidis et al., 2013).
NLRP3 forms a multiprotein inflammasome complex consisting of NLRP, pro-caspase 1, and the bipartite adaptor ASC (apoptosis-associated speck-like protein containing a carboxy-terminal CARD) (Lin et al., 2002). NLRP3 is expressed by myeloid cell types (monocytes, DCs, neutrophils and macrophages), as well as bronchial epithelial cells. The mechanism by which IAV activates the inflammasome is unclear, however, its importance has been proven using genetically modified mice lacking NLRP3. These mice have reduced viral-mediated inflammation in the lung, but increased mortality and viral clearance defects (Allen et al., 2009). Activation of inflammasomes results in the autocatalytic processing of pro-caspase 1 into its active form, which then cleaves pro-IL-1b and pro-IL-18 into IL-1b and IL-18, respectively (Wattrang et al., 2003). These activated cytokines then recruit monocytes and neutrophils into the lung, which are crucial to control infection and regulate tissue pathogenesis. In addition, the inflammasome participates in tolerance and tissue repair following an influenza virus infection (Iwasaki and Pillai, 2014). Furthermore, the role of NLRP3 in the protection against influenza virus is likely viral dose-dependent and maybe species-specific (Schaale et al., 2016).
Cytosolic RLRs are probably the most important group of PRRs during an IAV infection. This group of viral sensors consist of RNA helicase molecules and comprise RIG-I, Melanoma Differentiation-Associated protein 5 (MDA-5) and laboratory of genetics and physiology 2 (LGP2) (Pichlmair et al., 2006, Hornung et al., 2006).
RIG-I and MDA-5 recognize different types of RNAs absent in uninfected cells. RIG-I is most efficiently activated by short stretches of 5’ triphosphate (5’-ppp) or 5’ diphosphate (5’-pp) dsRNA, while MDA5 is activated by longer stretches of dsRNA in a 5’ phosphate-independent manner (Pichlmair et al., 2006, Yoneyama et al., 2004, Hornung et al., 2006, Loo et al., 2008). In the case of IAV, viral detection and IFN production in airway epithelial cells, DCs, and alveolar macrophages is RIG-I-dependent (Le Goffic et al., 2007, Pichlmair et al., 2006, Van Reeth, 2000). MDA-5 may still contribute to IFN induction by influenza viruses in certain hosts, such as chickens that lack RIG-I (Husser et al., 2011, Liniger et al., 2012).
Both RIG-I and MDA-5 contain two caspase activation and recruitment domains (CARDs) at their N-terminus, as well as a RNA helicase domain possessing dsRNA-dependent ATPase activity, and a C-terminal regulatory domain (CTD). Upon ligand binding, an ATP-dependent
conformational change occurs in RIG-I, leading to exposition of its two CARDs (Myong et al., 2009, Kowalinski et al., 2011). These domains are then ubiquitinated by E3 ligases TRIM25 (Gack et al., 2009) and RIPLET (Oshiumi et al., 2013), and/or bound to free poly-ubiquitin chains produced by TRIM25 (Zeng et al., 2010). Once activated, RIG-I can then interact with a mitochondrial adaptor that acts as an intermediate between detection of viral RNA and downstream activation events. This adaptor was discovered in 2005 by four different groups and was referred to as mitochondrial antiviral signalling protein (MAVS) (Seth et al., 2005), virus-induced signalling adaptor (VISA) (Xu et al., 2005), CARD adaptor inducing IFN-b (Cardif) (Meylan et al., 2005), and IFN-b promoter stimulator protein 1 (IPS-1) (Kawai et al., 2005). This adaptor leads to the oligomerization and scaffolding of RIG-I into a multi-kinase signalling platform that will activate NF-kB and the IFN regulatory factors, IRF3 and/or IRF7, to stimulate expression of IFNs (Osterlund et al., 2007, Paz et al., 2006)(Figure 1-4-A). LGP2 has a similar structure to RIG-I and MDA-5 but lacks the CARDs, and it has been proposed to function as a regulator of RIG-I and MDA-5 activity (Childs et al., 2013, Satoh et al., 2010) and IFN production (Rothenfusser et al., 2005). Indeed, LGP2 has been shown to potentiate IFN induction by synthetic mimic of viral dsRNA through co-operation with MDA-5. This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with MDA-5. In contrast, LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signalling (Childs et al., 2013).
Interestingly, dendritic cells from mice harbouring a point mutation of Lysine 30 to Alanine in LGP2 (Lgp2 (K30A/K30A)), that abrogated the protein’s ATPase activity, showed impaired IFN-b productions in response to various RNA viruses. Taken together, these data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain (Satoh et al., 2010). Finally, the level of LGP2 expression seems to be also critical in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low (Childs et al., 2013).
Figure 1-4: Type I IFN induction, signalling and action
(A) Induction of type I IFN. Single- and double-stranded (ss/ds)RNA, characteristic by-products of virus replication is recognised by RIG-I-like cytosolic sensors, RIG-I, MDA-5 and LGP2. Upon activation, RIG-I and MDA-5 will interact with a mitochondrial adaptor (IPS-1) and leads to activation of the transcription factors NF-kB, IRF-3/7 and c-jun/ATF-2 and formation of the enhanceasome. This complex recruits CBP and promote the formation of the basal transcriptional machinery and RNA polymerase II (not shown in this diagram), and allows full activation of the IFN-β promoter and production of IFN-β. Although not essential, formation of the enhanceasome is aided by HMGI. LGP2 has been proposed to function as a negative regulator of RIG-I and MDA-5 activity, and IFN-β induction. (B) Newly synthesized IFN-β binds to the type I IFN receptor (IFNAR) in a paracrine and/or autocrine manner. TYK2 then phosphorylates STAT2 (P-STAT2), while JAK1 phosphorylates STAT1 (P-STAT1). Following phosphorylation, P-STAT1 and P-STAT2 form a heterodimer that interacts with a monomer of Interferon Regulatory Factor 9 (IRF9). Together IRF9, P-STAT1 and P-STAT2 form the Interferon- stimulated gene factor 3 (ISGF3) complex. This complex is then transported into the nucleus, where it recognises a distinct DNA response element in the promoter of specific genes, the IFN-stimulated response element (ISRE). By binding to ISRE, the ISGF3 complex enhances transcription of hundreds of IFN-inducible or -stimulated genes (ISGs) that establish a cellular antiviral state (McBride et al. 2002). and activates the expression of numerous ISGs. Mx1, ISG15, OAS, PKR, TRIMs, IFITMs, CH25HC, tetherin, and viperin, among others are examples of induced proteins with antiviral activity. Furthermore, PRRs, IRFs, and several other signal transducing proteins, such as JAK, STAT1/2 and IRF9 are also induced by type I IFN. For details see text.
1.2.2. Natural Influenza PAMPs
It is likely that distinct PAMPs are generated at different stages of the virus life cycle. Several studies have indicated that incoming influenza A vRNPs are not sufficient to induce