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

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.


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].

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).

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 differentiationassociated 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 basepaired, 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 singlestranded 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.

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, doublestranded 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].

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].

NLRP3-Mediated Downstream Signaling
NLRP3-mediated signaling comprises five main components: NLRP3, apoptosisassociated 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].

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 IFNstimulated 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].

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).

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].

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 Cterminal 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].

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 (Figures 5-7).

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 (Figures 5-7).

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).

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'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), 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.

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.

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