Host Innate Antiviral Response to Influenza A Virus Infection: From Viral Sensing to Antagonism and Escape

Abstract

Influenza virus possesses an RNA genome of single-stranded, negative-sensed, and segmented configuration. Influenza virus causes an acute respiratory disease, commonly known as the “flu” in humans. In some individuals, flu can lead to pneumonia and acute respiratory distress syndrome. Influenza A virus (IAV) is the most significant because it causes recurring seasonal epidemics, occasional pandemics, and zoonotic outbreaks in human populations, globally. The host innate immune response to IAV infection plays a critical role in sensing, preventing, and clearing the infection as well as in flu disease pathology. Host cells sense IAV infection through multiple receptors and mechanisms, which culminate in the induction of a concerted innate antiviral response and the creation of an antiviral state, which inhibits and clears the infection from host cells. However, IAV antagonizes and escapes many steps of the innate antiviral response by different mechanisms. Herein, we review those host and viral mechanisms. This review covers most aspects of the host innate immune response, i.e., (1) the sensing of incoming virus particles, (2) the activation of downstream innate antiviral signaling pathways, (3) the expression of interferon-stimulated genes, (4) and viral antagonism and escape.

1. Introduction

Host innate immune system is the first line of defense against pathogens, including viruses. It encompasses physical and chemical barriers (e.g., skin and mucous), humoral innate molecules (e.g., lysozymes and cytokines), and cells (e.g., phagocytes) [1]. This system functions in two sequential stages: the sensing (afferent) stage and the effector (efferent) stage. The former is involved in recognizing the infection, while the latter is involved in responding and eliminating the infection [2]. The innate immune system in a host has three tasks: (1) recognize a diverse range of infecting pathogens, (2) respond to infection and kill or eliminate the pathogens, and (3) spare the host tissues while performing tasks 1 and 2 [2]. In addition, the innate immune system also contributes to the activation of the adaptive immune system [1]. Cytokines represent one of the most conserved components of innate immunity and spearhead the host innate immune response against viruses [1,3]. However, against viruses, tasks 2 and 3 of the innate immune response may not be as effective as against other pathogens. Viruses can effectively antagonize or evade the host innate immune response [4,5]. Furthermore, a hyperactive innate immune response can damage the host tissues and harm the host [6,7]. In this review, we have summarized tasks 1 and 2 of the cytokine-mediated host innate immune response to influenza virus infection and how the influenza virus antagonizes and escapes that response.

The influenza virus is the prototypic member of the family Orthomyxoviridae and is an enveloped virus with a segmented, linear, negative-sensed, single-stranded RNA genome [8]. Influenza virus exists in four types: A, B, C, and D. Influenza A virus (IAV) has a broad host range, infecting humans, other mammals, and various avian species. Influenza B virus (IBV) and influenza C virus (ICV) primarily infect humans. Influenza D virus (IDV) is the recently isolated influenza virus and is known to infect pigs and cattle [9]. IAV and IBV cause recurring seasonal epidemics in the human population [10,11]. IAV also causes occasional pandemics and sporadic zoonotic outbreaks in the human population [11,12]. The IAV undergoes regular inter-species transmission, particularly between humans, pigs, and avian species, and is the most diverse among influenza viruses [13]. Consequently, due to its relevance to human health, IAV is the most studied influenza virus and is the focus of this review.

The IAV genome is packaged as eight viral ribonucleoprotein (vRNP) complexes [14]. Each vRNP contains one of the eight RNA genome segments, nucleoprotein (NP), and three RNA-dependent RNA polymerase subunits: polymerase acidic (PA), polymerase basic 1 (PB1), and 2 (PB2). The eight vRNPs are surrounded by a host-derived lipid membrane (envelope), which is supported by an underlying layer of matrix 1 (M1) protein. The envelope is decorated with the receptor-binding protein, hemagglutinin (HA), a sialidase, neuraminidase (NA), and an ion-channel, matrix 2 (M2) protein [14]. To infect a host cell, IAV binds to the sialic acid receptor through HA and enters the cell via endocytosis. The viral envelope then fuses with the endosomal membrane, and the vRNPs are released inside the cytoplasm. The vRNPs are then trafficked to the nucleus, where viral RNA transcription and replication take place [14]. The viral mRNAs are exported to the cytoplasm where they are translated into up to 17 viral proteins—eight structural proteins (HA, M1, M2, NA, NP, PA, PB1, and PB2) and nine non-structural proteins (M42, NS1, NS2 or NEP, NS3, PA-X, PA-N155, PA-N182, PB1-F2, and PB1-N40) [15]. The NP, PA, PB1, and PB2 are imported back to the nucleus to assemble vRNPs. The M1 and NEP are also imported into the nucleus to facilitate the nuclear export of vRNPs, which are then trafficked to the plasma membrane [14]. The HA, NA, and M2 are directly transported to the plasma membrane via ER-Golgi transport [14]. At the plasma membrane, all viral components assemble into viral progeny, which are released from the cell by budding [14].

2. Sensing of IAV Infection by Host Cells

Host innate immune response is the first line of defense against virus infection, and the first stage of this is the sensing or detection of virus infection. Host cells sense virus infection via the pattern recognition receptors (PRRs). PRRs sense different stages of the virus infection in different intracellular compartments of different cell types. Multiple classes of PRRs are known, namely, Toll-like receptors (TLRs), retinoic acid-inducible gene-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), cyclic GMP-AMP synthase (cGAS), and Z-DNA-binding protein 1 (ZBP1). Human cells are known to employ TLRs, RLRs, NLRs, and ZBP1 to sense the IAV infection (Figure 1).

2.1. Toll-like Receptors (TLRs)

TLRs are germline-coded, membrane-bound proteins and expressed by both immune and non-immune cells. TLR genes were initially characterized in Drosophila embryo development [16,17] and its anti-microbial response [18]. Soon after, human homologs of Drosophila TLRs were identified and characterized for how they induce the innate immune response [19,20]. Subsequently, bacterial lipopolysaccharide (LPS) was discovered as the stimulant of TLRs [21,22,23,24,25]. Now, TLRs are known to sense various microbes, including viruses, through their specific molecular signatures, called pathogen-associated molecular patterns (PAMPs) [26].

TLRs are type I transmembrane proteins with an extracellular leucine-rich repeat (LRRs) for the detection of the PAMPs, a transmembrane domain for membrane insertion, and a cytoplasmic Toll/IL-1 receptor (TIR) domain for the downstream signaling [27]. Humans encode 10 TLRs (TLRs 1–10), which, based on their intracellular localization, are categorized into two groups. Group 1 comprises TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, which localize to the plasma membrane, and group 2 contains TLR3, TLR7, TLR8, and TLR9, which localize to the endosomes. The plasma membrane-localized TLRs recognize membrane-associated PAMPs, whereas the endosomal TLRs detect microbial nucleic acids, including of viruses [26,28].

IAV enters the human cells by endocytosis. Hence, IAV is sensed by TLR3 [29,30] through its double-stranded RNA, TLR7 [31,32,33,34], and TLR8 [33] through its single-stranded RNA (Figure 1). However, TLR4 also senses the IAV infection through damage-associated molecular patterns (DAMPs), which are released from IAV-infected cells [35,36]. Furthermore, TLR10 may also sense the IAV through a yet-to-be-known ligand [37] (Figure 1).

2.2. Retinoic Acid-Inducible Gene-like Receptors (RLRs)

RLRs are RNA helicases, which predominantly localize to the cytoplasm of most cell types. Three RLRs—retinoic acid-inducible gene-1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), are known. All RLRs are characterized by the presence of a central helicase domain and a C-terminal domain (CTD) for detecting the PAMPs. In addition, RIG-I and MDA5 possess two amino-terminal caspase activation and recruitment domains (CARDs) for downstream signaling [38]. RIG-I is the prototypic RLR and was first identified as one of the retinoic acid-inducible genes in differentiated leukemia cells [39,40]. Later, LPS was also found to induce the RIG-I expression [41]. Furthermore, a porcine homolog of RIG-I was found to be induced by the infection of porcine reproductive and respiratory syndrome virus (PRRSV) [42]. In 2004, Yoneyama et al. demonstrated that RIG-I senses double-stranded RNA (dsRNA) and induces the innate immune response against RNA viruses [43]. Subsequently, RIG-I was found to detect the dsRNA of multiple RNA viruses, including that of IAV [44,45]. Specifically, RIG-I senses short partially dsRNA strands containing base-paired, blunt-ended 5’ ends with a tri- or di-phosphate group, also known as a panhandle structure [46,47,48,49,50,51,52]. Such partially dsRNA panhandle structures in the life cycle of single-stranded RNA (ssRNA) viruses, like IAV, are generated during viral genome transcription and replication [53]. The MDA5 also senses similar but longer and virus-specific dsRNA architecture [45,51,54].

RIG-I has been shown to sense multiple forms of IAV replicating and transcribing RNAs through their panhandle-type architecture [47,52,55,56,57] (Figure 1). However, unlike most RNA viruses, IAV replicates its RNA genome in the host cell nucleus. Evidently, RIG-I is also localized to the nucleus [58] (Figure 1). There remains a scarcity of reports identifying MDA5 and/or LGP2 as the direct sensor of IAV RNA.

2.3. Z-DNA-Binding Protein 1 (ZBP1)

ZBP1 (also known as DAI) is the latest in PRRs and was identified as DLM-1 in tumor-activated macrophages [59]. DLM-1 was renamed as ZBP1 after the discovery that it possesses a Z-DNA-binding domain [60] and binds Z-DNA—a left-handed, double-stranded DNA helix [61,62]. A PRR function of ZBP1 was first reported when it was discovered to sense DNA as a PAMP and induce an innate immune response [63,64]. Later, ZBP-1 was also found to sense the RNA [65]. ZBP1 contains two N-terminal Z-nucleic acid binding domains (Zα1 and Zα2), a Z-DNA-binding domain (D3) next to Zα2, two central receptor-interacting protein homotypic interaction motif (RHIM) domains, and a C-terminal signal domain (SD) [60,66,67].

Like RIG-I, ZBP1 is mainly a cytosolic protein but is also localized to the nucleus [62,68,69,70]. ZBP1 senses the IAV infection by sensing multiple viral PAMPs—RNA, NP, and PB1 proteins as well as vRNP complex, both in the cytoplasm and in the nucleus [69,70,71,72,73] (Figure 1).

2.4. NOD-like Receptors (NLRs)

NLRs are also the recently described intracellular sensors of microbial PAMPs and DAMPs. The class II transactivator (CIITA) was the first NLR to be identified [74]. Now, 22 NLRs are known [75], and, phylogenetically, they are divided into three subfamilies: NOD, NLRP, and IPAF [76]. NLRs are characterized by the presence of N-terminal caspase recruitment (CARD) or pyrin (PYD) domain, a central nucleotide-binding and oligomerization domain (NACHT), and C-terminal leucine-rich repeats (LRRs) [76]. Upon sensing various PAMPs and DAMPs, NLRs oligomerize and form a multiprotein scaffold or platform, called the inflammasome [76]. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is the most characterized and significant among inflammasomes because it senses a diverse range of PAMPs and DAMPs [77].

Unlike TLRs and RIG-I, the NLRP3 inflammasome seems to sense the IAV infection through DAMPs rather than PAMPs. These DAMPs include (1) changes in the intracellular reactive oxygen species production [78] and ionic concentration [79], and lysosome function [78], (2) integrity or order of the Golgi complex [80] and viral proteins, such as PB1-F2 [81,82] and NP [83] (Figure 1). The sensing of NP is also facilitated by the host restriction factor MxA [83].

3. Downstream Innate Immune Signaling against IAV

3.1. TLR-Mediated Downstream Signaling

After detecting the PAMPs, TLRs signal via either myeloid differentiation primary response 88 (MyD88)-dependent pathway or Toll–interleukin 1 (IL-1) receptor (TIR)-domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent pathway.

In the MyD88 pathway, TLRs dimerize and recruit MyD88 via TIR domains. Then, MyD88 interacts with IL-1R-associated kinase 4 (IRAK4) through death domains (DDs) and activates the IRAK1 [84], forming a complex called the “Myddosome” [85]. Subsequently, IRAK1 activates the ubiquitin ligase, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) [86,87], which then ubiquitinates and activates the TGFβ-activated kinase 1 (TAK1) [87,88]. Activated TAK1 phosphorylates and activates the mitogen-activated protein kinases (MAPKs) and inhibitor of NF-κB (IκB) kinase (IKK) complex, which comprises IKKα, IKKβ, and NF-κB essential modulator (NEMO) [86,88,89]. The activated MAPKs then phosphorylate the transcription factor activator protein-1 (AP-1). On the other hand, the IKK complex phosphorylates interferon-regulatory factor (IRF) 5 and 7 as well as IκB. The phosphorylation of IκB leads to its ubiquitination and, subsequently, its proteasome-mediated degradation. This causes the release of NF-κB from IKK complex [90,91,92,93,94]. The phosphorylated AP-1, IRFs 5 and 7, and released NF-κB then translocate to the nucleus.

In the TRIF pathway, TLRs also dimerize and recruit TRIF, which then activates TRAF3 and TRAF6. Subsequently, TRAF3 interacts with TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) and inhibitor of κB (IκB) kinase-related kinase-ε (IKKε), which, in turn, phosphorylates the transcription factor IRF3. On the other hand, TRAF6 ubiquitinates receptor-interacting serine/threonine-protein kinase 1 (RIPK-1), which then phosphorylates TAK1. The result of these pathways is also the phosphorylation of AP-1 or IRFs, or release of NF-κB, and their subsequent translocation to the nucleus [95,96,97,98,99,100,101,102,103,104]. In the nucleus, AP-1, the IRFs, and NF-κB engage with their respective promoters and stimulate the expression of cytokines, interferons (IFNs), and pro-inflammatory cytokines.

As stated above, IAV infection is primarily sensed by the TLRs 3, 7, and 8. Both TLR 7 and 8 signal via the MyD88-dependent pathway, whereas TLR3 signals via TRIF-dependent pathway [26,28]. The canonical TLR3 [29,34,105,106,107,108,109] and TLR7 [31,32,33,105,110] signaling has been reported to occur during IAV infection (Figure 2). Furthermore, TLR4, which senses DAMPs from IAV-infected cells, also signals through the MyD88-dependent pathway [35] (Figure 2).

3.2. RLR-Mediated Downstream Signaling

RIG-I is present in an inactivated form in uninfected cells and in the absence of an RNA agonist. In this form, RIG-I is phosphorylated with sequestered CARDs [111,112,113,114,115,116]. Upon binding the viral RNA via CTD and helicase domain, RIG-I undergoes dephosphorylation and ATP-dependent conformational changes, which release the CARDs [116,117,118,119,120,121,122]. The CARDs then undergo sequential ubiquitination by multiple ubiquitin ligases, such as TRIM25 [123,124,125,126,127]. Such ubiquitination leads to the oligomerization and activation of RIG-I [128,129,130]. Activated RIG-I is then translocated to mitochondrial, peroxisomal, or mitochondrial-associated membranes, where it binds to mitochondrial antiviral signaling protein (MAVS) [131,132,133,134]. This interaction leads to the oligomerization and activation of MAVS [135]. Activated MAVS then forms a complex with TRAF3 (which ubiquitinates RIPK1), TNFR-associated death domain (TRADD) protein, and TANK to facilitate the activation and nuclear translocation of NF-κB and IRFs 3 and 7, which leads to the expression of IFNs α, β, or γ as well as pro-inflammatory cytokines [136,137,138]. The MDA5 gets activated and signals in a manner similar to RIG-I [111,121,130]. In contrast, LGP2, which binds dsRNA [139,140] but lacks CARDS, may sequester the dsRNA to antagonize RIG-I signaling [140,141,142,143]. However, LGP2 acts as a co-factor of MDA5 and promotes MDA5 signaling [54,139,143,144,145,146,147,148].

After sensing IAV infection, RIG-I signals via the canonical pathway described above, leading to the expression of IFNs and pro-inflammatory cytokines [34,45,47,101,106,129,149,150,151,152,153] (Figure 2). In addition to humans and mice, such anti-IAV RIG-I signaling has also been observed in ducks, waterfowl, and dogs [154,155,156,157,158] but not in chickens and turkeys, which lack RIG-I and some ubiquitin ligases [154,155,159]. Furthermore, NP of avian IAV H7N9 subtype can activate RIG-I signaling by binding directly to and stabilizing TRAF3 [160]. In addition, various co-factors, both proteins and non-coding RNAs (ncRNAs), such as E3 ligase FBXW7, deubiquitylase OTUB1, histone deacetylase 6 (HDAC6), RNA helicases DDX6 and DHX16, miRNA-136, lncRNA HCG4, and U1 snRNA, have been identified to augment RIG-I signaling in IAV-infected cells [161,162,163,164,165,166,167,168,169,170,171,172,173]. Interestingly, the infection-induced re-arrangement of the actin cytoskeleton can also activate the RIG-I and downstream signaling through the activation of protein phosphatase-1 and dephosphorylation of RIG-I [169].

MDA5 also signals through the RIG-I-like canonical pathway and induces the production of IFNs and pro-inflammatory cytokines in response to IAV infection in different host species [111,174,175,176]. In chickens that lack RIG-I, MDA5 is the main RLR to sense the IAV infection and induce the innate antiviral response [177,178]. In contrast, LGP2 seems to promote IAV infection by downregulating the innate antiviral response in infected cells [179,180].

3.3. ZBP1-Mediated Downstream Signaling

ZBP1 is an interferon-stimulated gene [63] and, upon binding the PAMPs or DAMPs, signals via multiple pathways, which leads to type I IFN expression and programmed cell death or PANoptosis [67]. In one pathway, ZBP1 is activated through phosphorylation by TANK-binding kinase 1 (TBK1) [181]. The activated ZBP1-TBK1 scaffold then recruits and phosphorylates IRF3, which then translocates to the nucleus and initiates type I IFN expression [63]. In another pathway, ZBP1 signals through the RIPK1–RIPK3 axis and activates NF-kB [64]. Additionally, ZBP1 signaling via RIPK1–RIPK3 axis also involves the caspase 8, which leads to PANoptosis—the caspase 8-mediated apoptosis, the NLRP3 inflammasome-mediated pyroptosis, and the RIPK3-MLKL-mediated necroptosis of cells [65,67,182].

During IAV infection, ZBP1 expression is upregulated in an IFN-dependent manner [69,183] (Figure 3). Furthermore, ZBP1 is ubiquitinated by TRIM34 and activated by the RIG-I-MAVS pathway [72,184] (Figure 3). Activated ZBP1 then senses various IAV PAMPs and DAMPs (as described above) and signals via the RIPK1–RIPK3–MLKL–caspase 8 axis and induces the PANoptosis of infected cells [69,70,71,185] (Figure 3). Caspase 6 is also an important cofactor in this axis [186]; however, it has been reported that this axis can also function independently of MLKL [187,188].

3.4. NLRP3-Mediated Downstream Signaling

NLRP3-mediated signaling comprises five main components: NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), interleukin (IL)-1β, IL-18, and caspase-1. The IL-1β, IL-18, and caspase-1 exist in an inactive “pro” form in unstimulated cells. NLRP3-mediated signaling is a two-step process. The first step is priming, where expression of NLRP3, pro-IL-1β, and pro-IL-18 is induced by pathways activated by other PRRs [189,190]. The second step is the formation and activation of the inflammasome, where, after sensing various DAMPs, NLRP3 oligomerizes and forms a complex with ASC. Then, pro-caspase-1 is recruited to this complex and becomes activated through self-cleavage. Subsequently, active caspase-1 cleaves pro-IL-1β and pro-IL-18 into active forms, which activate the pro-inflammatory and adaptive immune responses [191]. In addition, caspase-1 cleaves gasdermin D, which then triggers the pyroptosis of cells [189].

In IAV-infected cells, multiple mechanisms of NLRP3 inflammasome priming and activation have been described, with the involvement of several cofactors. It has been shown that IFN-inducible GTPase, IRGB10 [192], and transcription factor AP-1 [190] prime the NLRP3 inflammasome in IAV-infected cells (Figure 3). Furthermore, RIG-I activates NLRP3 inflammasome in IAV-infected cells by interacting with caspase-1 and ASC [193] (Figure 3). However, NLRP3 inflammasome can also be activated independent of RIG-I through the RIPK1–RIPK3–dynamin-related protein 1 (DRP1) axis [194,195] (Figure 3). Furthermore, ZBP1 can activate NLRP3 inflammasome in IAV-infected cells through the RIPK1–RIPK3–caspase-8 axis (Figure 3), where MLKL can be dispensable [69,187,188]. Nevertheless, both MLKL and caspase-8 can independently activate NLRP3 inflammasome in IAV-infected cells [188]. Therefore, it is plausible to say that, in IAV-infected cells, NLRP3 inflammasome is activated by the ZBP1–RIPK1–RIPK3–DRP1/caspase-8 axis. Furthermore, DEAD-box helicase 3 X-linked (DDX3X) [196], TRIM25 [197], and mitochondrial protein mitofusin 2 [198] augment the activation of NLRP3 inflammasome in IAV-infected cells.

3.5. IRFs in Downstream Signaling

IRFs are a nine-member family (IRFs 1–9) of transcription factors. All IRFs possess a conserved N-terminal DNA-binding domain with tryptophan repeats that bind to IFN-stimulated response elements (ISREs). The IRFs 3, 5, and 7 are most studied in the context of innate antiviral signaling and are involved in both RLR-mediated and TLR-mediated (MyD88-dependent and TRIF-dependent) downstream signaling [199]. After phosphorylation, IRFs dimerize and translocate to the nucleus, where they form a complex with histone acetyltransferases, such as p300/CBP [199,200,201,202,203]. The complex then binds to the ISREs in the promoter region of type I IFN genes and recruits an “enhanceosome” in order to initiate the transcription [204].

IRFs, 3, 5, and 7 have all been implicated in innate antiviral response during IAV infection [205,206,207,208,209,210] (Figure 2). Particularly, IRF3 and IRF7 seem to work in tandem [205,211] (Figure 2) and utilize annexin-A1 and cellular nucleic acid-binding protein (CNBP) as co-factors [212,213] for efficient activation and signaling to initiate the production of type I IFNs in IAV-infected cells. In addition, IRF1 has been shown to contribute to innate antiviral response during IAV infection [207,214,215]; one of the mechanisms for this is the positive regulation of ZBP1 expression and, consequently, ZBP1-mediated activation of NLRP3 inflammasome [215].

3.6. NF-κB in Downstream Signaling

NF-κB is a family of five (p50, p52, p65 or RelA, c-Rel, and RelB) transcription factors. All five possess a conserved N-terminal Rel homology region (RHR), which enables their dimerization and binding to DNA. Furthermore, RelA (p65), RelB, and c-Rel possess a C-terminal transactivation domain (TAD), which activates the transcription of their target genes [90]. After release from IKK complex, the NF-κB proteins dimerize and translocate to the nucleus. Here, NF-κB proteins bind to 9–11 base pair DNA nucleotide sequences, called κB sites, present in the promoter/enhancer region of various genes, including proinflammatory cytokines and chemokines, and initiate their transcription [216,217,218] (Figure 2).

NF-κB has been described to play a critical role in IAV-induced host innate immune response [219,220]. Furthermore, cofactors like FK506-binding protein 5 (FKBP5) [151] and Jade family PHD zinc finger 3 (JADE3) [221] enhance NF-κB signaling by enhancing either the release [222] or the nuclear translocation of NF-κB [221] in IAV-infected cells.

3.7. Interferons (IFNs)

IFNs are the main cytokines that combat viral infections. Three types of IFNs, I, II, and III, are known. Type I consists of IFN-α (multiple subtypes), IFN-β, IFN-ε, IFN-κ, and IFN-ω, type II consists of IFNγ, whereas type III consists of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4 [4]. Type I and III IFNs are expressed and secreted from infected cells via TLR- and RLR-mediated downstream signaling. Secreted IFNs then bind to infected and uninfected cells in an autocrine and paracrine manner, respectively, through their respective receptors, e.g., IFN alpha receptor (IFNAR) for type I IFNs and IFN lambda receptor (IFNLR) for type III IFNs [4]. This activates the Janus kinase (JAK)–signal transducer and activator of the transcription (STAT) pathway, resulting in the expression of hundreds of IFN-stimulated genes (ISGs), which create an “antiviral state” in IAV-infected cells [223,224]. Both type I and type III IFNs are produced by IAV-infected cells [211,225]. However, type III IFNs are the predominant IFNs in IAV-infected cells and are produced first, followed by the type I IFNs [226,227,228].

3.8. JAK-STAT Pathway

Cytokine receptors, JAKs, and STATs are three main components of the JAK-STAT pathway. The cytokine receptors are plasma membrane-localized membrane proteins with an extracellular domain, a transmembrane domain, and an intracellular domain. The JAKs are cytoplasm-localized protein kinases and exist in four types—JAK1, JAK2, JAK3, and TYK2. JAKs contain an N-terminal four-point-one, ezrin, radixin, and moesin (FERM) domain followed by the Src Homology 2 (SH2) and pseudokinase domains, and a C-terminal kinase domain. The STATs are transcription factors and localize to the cytoplasm in their inactive form. STATs exist in seven types—STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6—and contain several domains: an N-terminal domain (ND) followed by a coiled-coil domain (CCD), a DNA-binding domain (DBD), a linker domain (LK), a Src Homology 2 domain (SH2), and a C-terminal transactivation domain (AD). The cytokine receptors exist as dimers and remain associated with two molecules of JAKs through the interaction between their intracellular domain and JAKs’ FERM domain [229,230,231].

When cytokines, like IFNs, engage with their receptors, the two molecules of JAKs are auto activated by trans-phosphorylation. Activated JAKs then phosphorylate the intracellular domains of cytokine receptors, which creates the docking sites for STATs in JAK-cytokine receptor complex. Docked STATs are then phosphorylated by JAKs and dissociate themselves from the complex. The dissociated phosphorylated STATs then form a homodimer or heterodimer [230], which, in turn, binds IRF9 and forms the IFN-stimulated gene factor 3 (ISGF3) complex [232]. The ISGF3 then translocate to the nucleus, where it binds to ISREs of ISGs and initiates their transcription [229,230].

Above canonical JAK-STAT pathway, with the individual involvement of STAT1, STAT2, or STAT3 [233,234,235,236], has been described to be activated in IAV-infected cells, producing many ISGs [224] (Figure 4). However, the STAT1-mediated, STAT2-mediated, or STAT3-mediated antiviral response can also be activated in IAV-infected cells independent of cytokine receptor signaling via the RIG-I-MAVS axis [234,235,236] (Figure 4).

3.9. ISGs

Many ISGs, including proteins and ncRNAs, have been identified to express in IAV-infected cells (Figure 4) and described to inhibit IAV infection at different stages of its life cycle [224]. The ISGs, MUC1, and SPOCK2 inhibit the attachment of IAV virus particles to the host cell surface. The ISGs, hGBP-2, hGBP-5, IFITM 1, 2, and 3, NCOA7, RABGAP1L, and Serpin 1 inhibit IAV entry to host cells. The ISGs, CEACAM1, miRNA101, hGBP-3, p21, SERTAD3, IFIT 1, 2, and 3, ISG20, OAS 1, 2, 3, and L, TRIM 25 and 56, MOV10, MxA, PKR, and ZAPS inhibit the synthesis of either viral mRNAs or viral proteins, whereas the ISGs, ISG15, TRIM 14, 21, 22, and 35, and ZAPL target various host and viral proteins and inhibit IAV assembly. The ISGs, tetherin, and viperin inhibit IAV release. Finally, the ISGs, IFI16, miRNA485, circVAMP3, ISG15, lncRNAs, and SLFN 11 and 14 promote different stages of the host innate antiviral response against IAV [224].

3.10. Pro-Inflammatory Cytokines, Chemokines, and Growth Factors

Pro-inflammatory cytokines such as IL-1 and IL-18, chemokines such as IL-6, and growth factors such as G-CSF are produced through NF-κB signaling [218]. These molecules protect the lungs from IAV-induced inflammation, promote IAV clearance from the lungs, and increase host survival [237,238,239,240,241]. Furthermore, IL-1 and IL-18 promote a host-adaptive immune response against IAV [191,241,242,243]. However, the hyperactivation of these molecules during IAV infection, particularly under pre-existing medical conditions [244] or infection with an avian IAV [245], can increase lung injury and disease severity [242,246].

4. Antagonism of Host Innate Immune Response by IAV

Even though the host employs a multipronged innate antiviral response to restrict or eliminate the virus infection, viruses have evolved their own effective strategies to antagonize such response and multiply. These strategies include sequestration, degradation, downregulation of the expression, and interference in the function of the components of innate immune response. As described above, the host employs multiple pathways to exert a concerted antiviral response to restrict or eliminate IAV infection. In turn, IAV has evolved multiple mechanisms through the deployment of either viral or host proteins or host ncRNAs to effectively antagonize those host innate antiviral pathways (Figure 5, Figure 6 and Figure 7).

4.1. Antagonism of RIG-I

IAV antagonizes RIG-I-mediated sensing and signaling by multiple mechanisms and by employing both viral and host proteins (Figure 5).

IAV primarily employs non-structural 1 (NS1) protein, its main virulence factor, to antagonize the RIG-I-mediated signaling at multiple stages and by multiple mechanisms (Figure 5). NS1 is a multi-functional protein, possessing an N-terminal RNA-binding domain (RBD) followed by a linker region (LR), an effector domain (ED), and a C-terminal tail (CTT) [247]. Firstly, NS1 downregulates the expression of RIG-I in infected cells (Figure 5), either directly or indirectly, by (1) binding to RIG-I pre-mRNA and interfering with its maturation [248], (2) promoting the recruitment of the transcriptional repressor, CCAAT/enhancer binding protein beta (C/EBPβ) to RIG-I promoter [249], and (3) upregulating the expression of the RNase, monocyte chemotactic protein-induced protein 1 (MCPIP1) [250]. Secondly, NS1 interacts with RIG-I and competes for viral RNA binding, consequently blocking the viral RNA sensing [47,149,251,252,253,254] (Figure 5). Furthermore, NS1 interacts with and sequesters the ubiquitin ligases, TRIM25 and Riplet (Figure 5), blocking the RIG-I ubiquitination and, consequently, its activation [255,256,257,258]. NS1 also blocks RIG-I-mediated signaling further downstream. NS1 sequesters the RIG-I co-factors 14-3-3ε, OTUB1, and signaling molecule TRAF3 and interferes with the translocation of RIG-I to mitochondrial membrane [164,259] and the formation of MAVS–TRAF3 complex [260,261] (Figure 5), respectively.

PB1-F2 is another non-structural protein that IAV employs to antagonize the RIG-I signaling. PB1-F2 is a small protein encoded by +1 open reading frame of the IAV PB1 gene segment and localizes to the mitochondria [262,263]. Through localization to mitochondria, PB1-F2 binds MAVS and impairs downstream RIG-I signaling [262,263,264,265,266,267] (Figure 5). In addition, PB1-F2 causes the mitochondrial fragmentation and mitophagy [263,268].

IAV employs auxiliary functions of three RNA polymerase subunits—PA, PB1, and PB2—to antagonize the RIG-I signaling. For this, PA, PB1, and PB2 target MAVS (Figure 5) and either sequester it or promote its degradation [269,270,271]. Further, PA and PB1 can directly bind to and antagonize the RIG-I [272].

In addition, IAV recruits host proteins and ncRNAs such as K-homology-splicing regulatory protein (KHSRP) [273], an adenosine deaminase acting on RNA (ADAR1) isoform [274] and lncNSPL [275] to prevent the RIG-I activation, and miR340 [276] and T-cell immunoglobulin and mucin protein-3 (Tim-3) [277] to downregulate the RIG-I expression in infected cells (Figure 5). Furthermore, IAV degrades host HDAC6 [278] and progesterone receptor membrane component-1 (PGRMC1) [279], which promote RIG-I activation [173,279].

4.2. Antagonism of IRFs and NF-κB

Multiple IAV proteins antagonize IRF3 and IRF7. NS1 interacts with both IRF3 and IRF7 and inhibits their activation [280,281]. The NS2 protein (now known as nuclear export protein or NEP) of IAV also interacts with IRF7 and blocks its nuclear translocation [282] (Figure 5). Furthermore, viral RNA polymerase subunit, PA interacts with IRF3 and inhibits its activation [283] (Figure 5).

NS1 antagonizes NF-κB by interacting with IKK complex and blocking the degradation of IκB and release of NF-κB [284,285] (Figure 5). Furthermore, NS1 competes with NF-κB for binding to IFN λ promoter [286] (Figure 5). Also, PB1-F2 interferes with the binding of NF-κB to DNA [220] (Figure 5). PA-X, another IAV non-structural protein encoded by the PA gene segment through ribosomal frameshifting [287], antagonizes NF-κB by blocking its nuclear translocation [288] (Figure 5). In addition, IAV recruits the guanylate-binding protein 7 (GBP7) to inhibit NF-κB signaling in infected cells [289] (Figure 5).

4.3. Antagonism of NLRP3

IAV employs NS1 to antagonize the NLRP3-mediated signaling. NS1 interacts with NLRP3 and prevents NLRP3 inflammasome activation and, consequently, caspase 1 and IL-1β activation [290,291,292,293]. Furthermore, NS1 inhibits NLRP3 inflammasome activation by targeting the ASC [294] and TRIM25 [197] (Figure 6).

IAV also antagonizes the NLRP3-mediated signaling via PB1-F2, which interacts with NLRP3 and interferes with the NLRP3 inflammasome activation [295,296,297] (Figure 6).

4.4. Antagonism of JAK-STAT Pathway

IAV antagonizes the JAK-STAT pathway by multiple mechanisms. Firstly, NS1 upregulates the expression of host proteins, suppressor of cytokine signaling (SOCS) 1 and 3 [298]; SOCS1 and SOCS3 possess the inherent ability to inhibit JAK-STAT pathway in IAV-infected cells by targeting the JAKs and STATs [299,300] (Figure 7). Secondly, IAV PB2 and membrane protein HA promote the degradation of JAK1 [301] and type I and II IFN receptors [302,303], respectively (Figure 7). Thirdly, IAV exploits host microRNAs to interfere with the JAK-STAT pathway (Figure 7). IAV downregulates the expression of miR-30, which suppresses SOCS1 and SOCS3 expression [304]. Furthermore, IAV employs miR-93 and put-miR-34 to downregulate the expression of JAK1 [305] and STAT3 [306], respectively (Figure 7).

4.5. Antagonism of ISGs

In addition to antagonizing the upstream innate immune pathways, IAV directly antagonizes some of the ISGs by multiple mechanisms, and here too, IAV employs NS1 protein. NS1 competes with ISGs, PKR, and OAS (Figure 7) for binding to viral dsRNA PAMP and blocks the activation of PKR-mediated pathway, which inhibits viral protein synthesis [307,308,309] and RNase L, which degrades viral RNA [310], respectively. Furthermore, NS1 interacts with hGBP-1 (Figure 7) and inhibits the GTPase activity required for its antiviral function [311]. To antagonize tetherin, IAV employs its membrane protein, M2 which interacts with tetherin (Figure 7) and promotes its degradation [312]. Finally, IAV recruits the ubiquitin ligase, NEDD4 [304,313] and methyltransferase, SET7 [314] (Figure 7) to ubiquitinate and methylate IFITM3, respectively, and decrease its antiviral activity.

5. IAV’s Escape from the Host Innate Immune Response

IAV can escape the innate antiviral response in hosts carrying the genetic variants of antiviral factors due to single-nucleotide polymorphisms (SNPs) or mutations (

Table 1. Host gene variants allowing IAV to escape the innate immune response.

Genes	Variants/Mutations	Effect
TLR3	rs5743313/CT, rs5743313, L412F, P554S/P680L, rs3775291/rs3775290	Escape from TLR3-mediated signaling
RIG-I (DDX58)	p.R71H/p.P885S, rs4487862	Escape from RIG-I-mediated signaling
IRF7	p.Phe410Val (F410V)/p.Gln421X(Q421X), E331V	Escape from weakened IFN response
IRF9	c.991G > A	Escape from weakened IFN response
MxA	Mutations in GTPase domain	Escape from MxA-mediated restriction
IFITM3	rs12252-C	Escape from IFITM3-mediated restriction
OAS	rs10774671	Escape from OAS-mediated restriction
Serpin 1	rs6092	Escape from Serpin 1-mediated restriction

), which reduce their antiviral activity and weaken the innate immune response. Furthermore, IAV can escape the host restriction by acquiring mutations in viral targets of antiviral factors.

5.1. Escape from TLR- and RLR-Mediated Sensing

Multiple variants of TLR genes have been reported in humans. IAV escapes the TLR-mediated sensing and causes severe disease in humans carrying some of those variants [315,316,317,318,319,320]. IAV infection leads to pneumonia or acute respiratory distress syndrome (ARDS) in humans carrying the SNPs rs5743313/CT [315], rs5743313 [316], L412F [318], P554S/P680L [317,319], and rs3775291/rs3775290 [320] in TLR3 gene (Table 1).

Similarly, IAV escapes the RIG-I-mediated sensing and downstream signaling in humans carrying the SNPs p.R71H/p.P885S [321] and rs4487862 [322] in RIG-I (DDX58) gene and causes severe disease (Table 1).

5.2. Escape from IRF-Mediated Signaling

The variants of IRF7 and IRF9 genes have also been reported in humans [323,324,325,326,327]. IAV escapes the weakened IFN response and causes severe disease in humans carrying the IRF7 variants p.Phe410Val (F410V)/p.Gln421X(Q421X) [324] and E331V [326] and IRF9 variant c.991G > A [325] (Table 1).

5.3. Escape from ISG-Mediated Restriction

The ISG MxA is a GTPase and restricts IAV infection by sequestering the vRNPs [224]. IAV escapes the MxA-mediated restriction in humans carrying Mx1 gene variants, which encode for MxA deficient in GTPase activity [328,329,330] (Table 1). Human MxA is also a barrier against the transmission of zoonotic IAVs to humans [224]. However, human IAV have acquired the mutations in their NP to escape the MxA-mediated restriction in human cells [331,332,333]. Similarly, zoonotic IAV escapes the human MxA barrier by acquiring human-adaptive mutations in their NP [334,335,336,337].

IFITM3 is another prominent ISG that localizes to the endosome and restricts IAV infection by interfering with its endocytic entry to host cells [224]. IAV escapes the IFITM3 restriction and causes severe disease in humans [338,339], mainly from the Asian ethnicity [316,340,341,342,343,344,345,346,347], carrying the IFITM3 gene variant rs12252-C. This variant encodes N-terminally truncated IFITM3, which is unable to localize to the endosome, and hence, allows IAV to escape the IFITM3 restriction [338,348,349,350] (Table 1). Furthermore, avian IAVs may escape the IFITM3 restriction in human cells with ineffective endosomal acidification [351].

Similarly, IAV escapes the restriction imposed by ISGs, OAS, and Serpin 1 in humans carrying OAS1 gene variant rs10774671 [352] and Serpin 1 gene variant rs6092 [353], respectively (Table 1).

6. Summary

The host employs multiple innate immune pathways and a complex network of factors in each pathway to sense, block, and eliminate the IAV infection. However, IAV employs its own proteins, notably NS1, as well as recruits host factors, both proteins and ncRNAs, to antagonize multiple steps of the host innate immune response.

IAV continues to pose a significant burden on global public health annually through seasonal epidemics [354]. Furthermore, the threat of another IAV pandemic is real as exemplified by the increased incidents of zoonotic infections, evolution, and cross-species transmission of avian IAVs, particularly the H5N1 subtype [12]. Alarmingly, an avian IAV H5N1 subtype has been discovered recently to infect dairy cows—a previously unknown host to IAV [355]—and associated dairy farm workers [356,357]. Three classes of antiviral drugs—M2 ion channel inhibitors, NA inhibitors, and PA inhibitors—are available to treat IAV infections. However, these drugs are prone to be ineffective over time because they target the IAV components M2, NA, and PA, respectively, and IAV can mutate these components to acquire resistance to these drugs [358,359]. Indeed, the M2 ion channel inhibitors are not recommended for the treatment of IAV infections anymore, because the majority of circulating IAV strains have acquired resistance to them [360]. Therefore, a comprehensive knowledge of the interplay between host innate immune response and IAV is crucial to designing alternative antiviral therapies targeting the host factors involved in innate immune response [361,362].

Indeed, some of the knowledge gained in this space has already been applied, e.g., the use of TLR agonists for treating IAV infections [363] or as adjuvants in flu vaccine formulations [364]. Furthermore, inhibitors of critical components of the innate pathways, e.g., RIPK3 in ZBP1-mediated necroptosis [365], are being developed to prevent the severity of IAV disease.