Innate Immune Evasion Strategies of Influenza Viruses

Benjamin G Hale; Randy A Albrecht; Adolfo García-Sastre

Future Microbiol. 2010;5(1):23-41.

Host Innate Immune Response to Infection

Interferon from choriontic membranes of embryonated chicken eggs was first noted as a naturally produced antiviral substance during the middle of the 20th century by Isaacs and Lindenmann.^[31,32] Since its initial discovery, three IFN types have been identified,^[33] including type I IFN (mainly α/β), type II IFN (γ) and type III IFN (λ). Type II IFN contributes to the establishment of adaptive immune responses.^[34,35] Although type III IFN has been shown by several studies to control influenza virus infection,^[36] the type I IFN response appears most critical for limiting influenza virus replication and thereby ensuring host survival.^[37–41]

Type I IFN Induction

Virus replication results in the synthesis of several types of pathogen-associated molecular patterns (PAMPs). The major influenza virus PAMP is thought to be cytoplasmic viral RNA species that contain triphosphate groups at their 5′ ends (as opposed to the 7-methyl guanosine cap structures present at the 5′ ends of cellular transcripts).^[42] dsRNA is a widely recognized PAMP produced during infection with some RNA viruses. Although influenza viruses do not appear to produce detectable amounts of dsRNA during infection,^[42,43] low levels of viral dsRNA might also represent an influenza virus PAMP. Host cells detect the presence of an infecting virus by pattern recognition receptors (PRRs) that recognize the PAMPs and initiate antiviral signaling cascades that ultimately effect an antiviral response (reviewed in ^[13]). PRRs are divided into several families, including nucleotide-binding oligomerization domain (NOD)-like receptors, Toll-like receptors (TLRs), and retinoic acid-inducible gene-I (RIG-I)-like helicases (reviewed in ^[44]). Since the initial observation that virus infection induces caspase-1 activation in a cryopyrin/Nalp3-dependent manner,^[45] the potential contribution of NOD-like receptors to the establishment of innate immune responses against influenza virus infection has been more closely examined,^[46–48] but this will not be discussed further here.

Toll-like receptors are the most extensively studied family of PRR. They are transmembrane proteins expressed by multiple cell types and are located on either the cell surface (TLR1, 2, 4 and 5), or on cytoplasmic structures such as endosomes (TLR3, 7, 8 and 9; reviewed in ^[49]). Although most surface TLRs recognize bacterially derived PAMPs, TLR4 has been shown to recognize the fusion glycoprotein of respiratory syncytial virus.^[50] It is unknown whether TLR4 also recognizes either of the two influenza virus surface glycoproteins (hemagglutinin and neuraminidase [NA]), but TLR4 signaling during infection with highly pathogenic H5N1 influenza A virus has been reported to contribute towards lung pathology.^[51] TLRs are generally necessary for antigen-presenting cells (i.e., macrophages and plasmacytoid dendritic cells) to respond to virus infection by inducing innate immune responses.^[49] In particular, TLR7 is critical for influenza virus-induced IFN production by plasmacytoid dendritic cells.^[52,53]

Retinoic acid-inducible gene-I-like helicases are present in the cytoplasm of many cell types, including conventional dendritic cells, macrophages and pulmonary epithelial cells (the principle target for influenza virus replication).^[54] The RIG-I-like helicase family is primarily comprised of the DExDH box RNA helicases RIG-I, melanoma differentiation-associated gene-5 (MDA-5), and laboratory of genetics and physiology-2 (LGP-2). These PRRs are thought to 'sense' distinct viral cytoplasmic RNA PAMPs produced during infection, and may act either antagonistically or in synergism with one another (reviewed in ^[13]). Nevertheless, studies with cells and mice deficient in either RIG-I or MDA-5 suggest that only RIG-I is essential for induction of IFN in response to influenza virus infection.^[55–58]

Since the first observation of the critical contribution of RIG-I in inducing innate immunity,^[59] a detailed picture of the signaling pathway that leads from RIG-I activation to induction of IFN has emerged (summarized in Figure 1). RIG-I is comprised of several functional domains, including two tandem amino-terminal caspase activation and recruitment domains (CARDs), a central ATP-dependent helicase domain and a carboxyl-terminal regulatory domain (RD).^[56] In its inactive state, RIG-I exists as a monomer in which the RD is thought to mask the CARDs. Binding of the RD to PAMPs (either dsRNA or cytoplasmic 5′-triphosphate containing viral RNA) results in a conformational change leading to RIG-I dimerization and exposure of the CARDs (reviewed in ^[60]). Gack et al. recently demonstrated that ubiquitination of lysine 172 within the second CARD of RIG-I is essential for IFN production in response to virus infection.^[61] This posttranslational modification of RIG-I is performed by the IFN-inducible E3 ubiquitin ligase, tripartite motif (TRIM)25.^[61] Following ubiquitination, RIG-I initiates a signaling cascade that begins with its relocalization to mitochondria, where the exposed, ubiquitinated CARDs of RIG-I associate with the CARD of mitochondrial antiviral signaling adaptor (MAVS; also known as IPS-1/VISA/Cardif).^[62] MAVS functions as an essential scaffolding factor^[63] that recruits two multiprotein 'signalosome' complexes consisting of a variety of E3 ubiquitin ligases, additional scaffolding proteins and numerous protein kinases (for recent reviews see ^[64,65]). The first complex contains TNF-receptor-associated factor 3 (TRAF3), TRAF family member-associated NF-κB activator (TANK), TBK1, and IKKε, which phosphorylates the transcription factor IFN regulatory factor 3 (IRF-3). The second kinase complex consists of TRAF6, receptor-interacting protein (RIP)1, NF-κB essential modulator (NEMO), TAK1, IKKα and IKKβ, which phosphorylates inhibitor of κB (IκB), ultimately leading to NF-κB activation. Phosphorylated IRF-3, activated NF-κB and ATF-2/c-Jun all translocate to the nucleus, where they form an enhanceosome complex on the IFN-β promoter and transcribe IFN-β mRNA (Figure 1) (reviewed in ^[13]).

(Enlarge Image)

Figure 1.

Retinoic acid-inducible gene-I-mediated type I interferon pathway and its regulation during influenza A virus infection. (A–E) Critical checkpoints. (A) Virus replication produces triphosphorylated vRNA and potentially dsRNA byproducts that are pathogen-associated molecular patterns recognized by cytoplasmic RIG-I. (B) NS1 regulates the activation of RIG-I by binding to and sequestering dsRNA and/or by interaction with RIG-I. Formation of viral RNP complexes may also contribute to 'hiding' pathogen-associated molecular patterns from RIG-I. (C) Binding of NS1 to TRIM25 prevents essential ubiquitination of RIG-I. (D) Cap-snatching activity of the viral polymerase complex may reduce the pool of host antiviral mRNAs available for nuclear export and translation. The NS1 protein also directly inhibits global cellular pre-mRNA processing by binding to host CPSF30. (E) NS1 binds to components of the nuclear pore complex and inhibits nuclear export of cellular mRNA.
CPSF30: 30-kDa cleavage and polyadenylation specificity factor; IRF: Interferon regulatory factor; MAVS: Mitochondrial antiviral signaling adaptor; NEMO: NF-κB essential modulator; NS1: Nonstructural protein 1; PPP: Triphosphate; RIG: Retinoic acid-inducible gene; RIP: Receptor-interacting protein; RNP: Ribonucleoprotein; TANK: TRAF family member-associated NF-κB activator; TRAF: TNF-receptor-associated factor; TRIM: Tripartite motif; Ub: Ubiquitin.

Type I IFN Signaling

After translation, newly synthesized bioactive IFN-β is secreted from the infected cell and engages with either the IFN-α/β receptor (IFN-α/βR) of the same cell (autocrine signaling) or the neighboring cell (paracrine signaling). The IFN-α/βR is a dimeric structure composed of the subunits IFN-α/βR1 and IFN-α/βR2; the cytoplasmic tails of these subunits serve as docking platforms that initiate a signaling cascade in response to IFN-β (Figure 2). This cascade is dependent on coordinated protein phosphorylation and protein–protein interactions involving Tyk2, Jak1, STAT1 and STAT2.^[66] The consequence of this signaling cascade is the generation of a nuclear IFN-stimulated gene factor-3 transcription factor complex (ISGF3), which comprises a heterodimer of phosphorylated STAT1/STAT2 complexed with IRF-9 (reviewed in ^[13]). Activated ISGF3 stimulates the transcription of over 300 genes that lie downstream of IFN-stimulated response elements^[67] (reviewed in ^[66]). These gene products establish a general 'antiviral state' within cells that limits virus replication (Figure 2). For influenza viruses, the best-characterised antiviral proteins include: dsRNA-activated protein kinase, PKR (translational repression^[68]); 2′–5′ oligoadenylate synthetase (OAS; activator of RNaseL, mRNA degradation^[69]); myxovirus resistance gene A (MxA; dynamin-like large GTPase recognising and inhibiting the viral ribonucleoprotein [RNP] structure^[70]); viperin (inhibits viral release^[71]); and IFN-stimulated gene (ISG)15 (a ubiquitin-like modifier that apparently regulates a number of IFN-stimulated proteins^[72,73]).

(Enlarge Image)

Figure 2.

Type I interferon receptor signaling pathway and expression of interferon-stimulated genes. Binding of IFN-α/β to the IFN receptor stimulates the phosphorylation of STAT1 and STAT2. Phosphorylated STAT1 and 2 associate with IRF-9 to form the transcription factor ISGF3, which relocalizes to the nucleus and stimulates the transcription of ISGs whose promoters contain IFN-stimulated response elements. Although IFN stimulates the transcription of more than 300 ISGs, only the antiviral functions of a small percentage have been well characterized. Notable examples of IFN-induced antiviral effectors and their regulation by influenza viruses are indicated. See text for further details.
A/NS1: Influenza A virus NS1 protein; B/NS1: Influenza B virus NS1 protein; IFN: Interferon; IRF: IFN regulatory factor; ISG: IFN-stimulated gene; MxA: Myxovirus resistance gene A; NP: Nucleocapsid protein; NS1: Nonstructural protein 1; OAS: 2′-5′ oligoadenylate synthetase; SOCS: Suppressor of cytokine signaling.

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Abstract and Introduction
Influenza Viruses
Host Innate Immune Response to Infection
Influenza Virus Antagonism of Innate Immunity
Future Vaccines & Antivirals
Future Perspective
References
Sidebar

Table 1. The 11 proteins encoded by influenza A viruses, their functions and potential antiviral susceptibility.
Protein	Segment	kDa	Major Function(s)	Antiviral targets	Ref.
HA	4	64	Attachment to host cells; fusion of viral and host membranes	Fusion inhibitors (e.g., TBHQ) Sialic acid removal (e.g., recombinant sialidase DAS181)	^[200] ^[201,202]
NA	6	50	Cleavage of sialic acid; release of virions from cells	Neuraminidase inhibitors (oseltamivir^‡, zanamivir^‡ and CS-8958*)	^[203]
PB1	2	87	Polymerase component	PB1–PA interaction Polymerase function (e.g., T-705*)	^[204] ^[205]
PB2	1	86	Polymerase component	Cap binding	^[116,172]
PA	3	83	Polymerase component	PB1–PA interaction Endonuclease	^[204] ^[118,119]
NP	5	56	RNA encapsidation; RNP component	RNA binding? Oligomerization pocket?	^[172]
M1	7	28	Matrix protein; structural support of virion	Oligomerization?
M2	7 (spliced)	11	Ion channel; dissociation of viral components during uncoating	Ion channel inhibitors (amantadine^‡ and rimantidine^‡).
NS1	8	26	Antagonism of host immune responses	RNA binding, CPSF30 binding, IFN antagonism*	^[187] ^[105] ^[178]
NEP	8 (spliced)	14	Nuclear export of RNPs	Sites of RNP interaction?
PB1-F2	2 (+1 ORF)	8	Induction of apoptosis in immune cells	Sites of interaction with cellular proteins? Oligomerization?

For each protein, its corresponding RNA genomic segment is indicated, as well as its approximate molecular weight (in kDa).
*Compounds identified with antiviral activity in experimental trials.
^‡US FDA-approved antivirals.
CPSF30: 30-kDa cleavage and polyadenylation specificity factor; F2: Frame 2; HA: Hemagglutinin; IFN: Interferon; M1: Matrix protein 1; NA: Neuraminidase; NEP: Nuclear export protein; NP: Nucleocapsid protein; NS: Nonstructural; ORF: Open reading frame; PA: Polymerase subunit acidic; PB: Polymerase subunit basic; RNP: Ribonucleoprotein; TBHQ: Tert-butyl hydroquinone.

Box 1. Influenza A viruses.
Influenza A virus particles (virions) have a typical diameter of 80–160 nm, and are pleomorphic in shape. Virions consist of a lipid envelope (derived from the host plasma membrane) out of which protrude two surface glycoproteins: hemagglutinin and neuraminidase. There are 16 different hemagglutinins and nine different neuraminidases, and reassortment of these two antigens determines the influenza A virus subtype (e.g., H1N1 or H3N2). Embedded within the lipid envelope bilayer is the viral integral membrane ion channel protein, M2, whilst the viral matrix protein (M1) lies underneath the envelope and structurally supports the particle. Contained within this matrix shell is the viral RNA genome, which (for influenza A viruses) consists of eight single-stranded, negative-sense RNA segments (negative-sense refers to the genome being complementary to mRNA, which by convention is termed positive-sense). Each strand is wrapped up in multiple copies of the nucleocapsid protein, and is associated with three proteins responsible for transcription and replication: polymerase subunit basic (PB)1, PB2 and polymerase subunit acidic (the viral polymerase complex). Each nucleocapsid protein-encapsidated RNA segment, together with its heterotrimeric polymerase, constitutes a viral ribonucleoprotein complex. Although consisting of only eight genomic segments, multiple gene-encoding mechanisms mean 11 viral proteins are produced during infection (reviewed in ^[1]). Other than the proteins detailed above, the nuclear export protein, nonstructural protein 1 and PB1-frame 2 are produced. Only nonstructural protein 1 and PB1-frame 2 are not incorporated into the virus particle, and are found only in infected cells. Functions of each viral protein are listed in Table 1.

Box 2. Antigenic drift versus antigenic shift.
Antigenic drift is the process whereby mutations accumulate in viral surface glycoproteins (predominantly hemagglutinin), resulting in viruses with slightly different antigenic profiles. Antigenic shift occurs when the surface hemagglutinin and neuraminidase segments reassort between different viruses, resulting in a virus with completely new surface glycoproteins.

Sidebar

Executive Summary

Influenza viruses

In humans, influenza virus infection is usually limited to the upper respiratory tract. In certain severe cases the lower respiratory tract can be affected, causing viral pneumonia often exacerbated by secondary bacterial infection.
'Seasonal influenza' is caused annually by new viruses possessing slightly different surface antigens. Such epidemics account for approximately 500,000 deaths worldwide.
Global influenza pandemics occur when antigenically novel influenza viruses emerge. The most devastating to date, in 1918, was responsible for over 40 million deaths.
In March 2009, a novel swine-origin H1N1 influenza A virus emerged from pigs into the human population and has spread to become the latest influenza pandemic.

Host innate immune response to infection

The interferon (IFN) system is a powerful innate immune defence used by cells to limit virus replication.
Viral pathogen-associated molecular patterns (PAMPs) are detected by host cell sensors (e.g., retinoic acid-inducible gene-I), which then initiate a complex signaling cascade resulting in the synthesis and secretion of IFNs.
IFNs can act in either an autocrine or paracrine manner to upregulate over 300 antiviral genes that ultimately lead to suppression of virus propagation.

Influenza virus antagonism of innate immunity

Influenza viruses have evolved numerous strategies in order to evade the host innate immune response.
The viral nonstructural (NS)1 protein acts at multiple levels to prevent IFN production: NS1 can limit PAMP detection, block essential retinoic acid-inducible gene-I post-translational modifications and block the processing and export of IFN mRNAs.
NS1 may also limit IFN responses by preventing expression of antiviral proteins or by directly inhibiting their activities.
Other strategies used by influenza viruses to evade the effects of IFN include: limiting PAMP formation, general inhibition of host cell protein synthesis, replication compartment, replication speed, induction/inhibition of apoptosis and reduced sensitivity to host antiviral proteins.

Future vaccines & antivirals

Vaccination is the best means of controlling both seasonal and pandemic influenza outbreaks. No 'universal' influenza vaccine exists; thus, new seasonal vaccines must be developed annually.
One strategy for vaccine design is to use influenza viruses engineered to express mutant NS1 proteins. The replication of such viruses is restricted in vivo; thus, they are good candidates for live-attenuated vaccines.
Antiviral drugs may be the 'first line of defence' whilst vaccines are generated to novel seasonal or pandemic viruses.
The development of viral resistance to existing licensed antiviral drugs is a cause for concern; thus, new antivirals targeting other viral components are urgently required.
Several sites on the influenza A virus NS1 protein may be targets for novel antivirals.
IFN and other agonists of host innate immunity may be useful as future therapeutics.

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