Innate Immune Evasion Strategies of Influenza Viruses

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

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

Influenza Virus Antagonism of Innate Immunity

The seminal study by Isaacs and Lindenmann^[31] revealed that treatment of choriontic membranes with heat-inactivated influenza virus stimulates the release of an inhibitory substance (IFN) that limits the replication of infectious influenza viruses. In hindsight, these studies also indicated that, unlike heat-inactivated virus, infectious influenza viruses do not efficiently stimulate IFN production. More recent in vitro studies using immortalized human lung cell lines have confirmed that, in general, wild-type influenza A viruses are poor inducers of type I IFN.^[74] Several further studies have uncovered the many strategies employed by influenza viruses to limit both directly and indirectly the global cellular antiviral state. As outlined below, this is thought to primarily involve viral targeting of the IFN-induction and signaling cascades at multiple levels. Influenza viruses are by no means unique in their ability to limit the IFN response, and in order to replicate efficiently all viruses must be able to counteract these host defences to some extent. How other viruses subvert innate immunity has been reviewed elsewhere.^[13,55,64]

Nonstructural Protein 1 Limits IFN Production

The influenza A virus nonstructural (NS)1 protein is a multifunctional virulence factor, the major function of which appears to be antagonism of host innate immunity (for an extensive recent review of the structure and functions of NS1 see ^[75]). This was first demonstrated after reverse genetics techniques allowed the generation of recombinant influenza viruses that either lacked the NS1 gene^[38] or expressed NS1 truncation mutants.^[76] Infection of cell cultures and animal models with these viruses revealed that the mutant viruses induced robust IFN secretion from infected cells.^[77–82] Furthermore, the importance of NS1-mediated IFN inhibition was confirmed when these mutant viruses were shown to cause significantly reduced morbidity in mice, chickens, swine, horses and macaques.^{[38,78–80,83–86]} Using a mouse model of infection, it has recently been proposed that NS1 expression allows for a brief period of 'stealth' virus replication preceeding the onset of host innate and adaptive immune responses.^[87] It should be noted that the IFN-antagonistic property of NS1 appears functionally conserved between both influenza type A and B viruses.^{[38,76,88,89]}

NS1 Inhibits the RIG-I Signaling Cascade

Initial in vitro and cell culture assays revealed that the NS1 protein prevents virus-induced activation and translocation of IRF-3,^[90] NF-κB^[91] and ATF-2/c-Jun.^[92] Thus, by blocking activation of these individual components of the enhanceosome complex, the NS1 proteins of both influenza A and B viruses can limit RIG-I-mediated transcriptional activation of the IFN-β promoter.^[42,93–95] Furthermore, microarray studies have revealed several classes of genes whose transcriptional activation is dependent on activated RIG-I^[56] or phosphorylated IRF-3.^[96] Thus, in addition to inhibiting IFN-β gene expression, the NS1 protein likely prevents expression of numerous other cellular genes that are also transcriptionally dependent on the RIG-I-mediated signaling cascade.

Biochemical studies initially suggested that IFN inhibition by the influenza A virus NS1 protein requires formation of a complex containing NS1, RIG-I and possibly a viral PAMP (e.g., dsRNA).^[42,95] Such a complex appears to allow NS1 to block activation of the IFN-β promoter even in the presence of a constitutively active version of RIG-I composed only of its CARDs.^[42,93–95] However, it is not clear whether NS1 can bind directly to RIG-I,^[95] and recent data have uncovered further mechanistic details about the nature of this multiprotein inhibitory complex: the NS1 proteins of human, avian and swine influenza A viruses interact with and inhibit the activity of the E3 ubiquitin ligase, TRIM25 (Figure 1),^[97] which (as described above) is required to posttranslationally modify RIG-I and thereby stimulate its signaling cascade.^[61]

Tripartite motif 25 retains several evolutionarily conserved domains that are common among the ten subfamilies in the TRIM family,^[98] including an amino-terminal 'really interesting new gene' (RING) domain that mediates its enzymatic function, central B-boxes and a coiled-coil domain that mediates oligomerization. However, TRIM25 possesses a carboxyl-terminal splA and ryanodine receptor (SPRY) domain that is not shared by all subfamilies of TRIM proteins.^[98] The SPRY domain of TRIM25 was shown to bind the CARDs of RIG-I.^[61] TRIM25-mediated ubiquitination of RIG-I in response to virus infection is a complex process involving coiled-coil domain-mediated oligomerization, binding of the SPRY domain to the first CARD domain of RIG-I,^[61,99] and ubiquitination of lysine 172 within the second CARD of RIG-I by the RING domain of TRIM25.^[61] NS1 was shown to prevent TRIM25 oligomerization by interacting with its coiled-coil domain, thereby preventing TRIM25 ubiquitinating RIG-I.^[97] The specificity by which the NS1 protein binds the coiled-coil domain of TRIM25 has not been completely examined and, therefore, whether NS1 interacts with additional members of the TRIM family has yet to be determined. In addition, given that proteins other than RIG-I appear to be substrates for both TRIM25-mediated ubiquitination^[100] and ISGylation,^[101] TRIM25 inhibition by NS1 may have a number of additional consequences during infection. Although it is known that the influenza B virus NS1 protein can also block IFN production pretranscriptionally,^[89] it is unclear if this NS1 also binds TRIM25. As detailed below, not all functions of the influenza A and B virus NS1 proteins are conserved.

Influenza Viruses Regulate Host Cell Gene Expression

In addition to directly blocking the cytoplasmic signaling cascade that results in IFN-β gene transcription, the influenza A virus NS1 protein has also been reported to deregulate general host cell gene expression.^[102] This is largely accomplished by NS1 binding two zinc-finger domains of the nuclear 30-kDa subunit of cleavage and polyadenylation specificity factor (CPSF30).^[102–105] This interaction prevents CPSF30 from carrying out its normal cellular function of correctly processing the 3′ ends of pre-mRNAs into mature, polyadenylated mRNAs (Figure 1).^{[102,106–108]} Biochemical and crystallographic studies of the NS1–CPSF30 complex revealed the regions of NS1 necessary for its interaction with CPSF30.^[103,109] The main interaction site is centered around tryptophan-187, whilst residues including phenylalanine-103 and methionine-106 contribute to stability of the NS1–CPSF30 complex.^[103,109] These insights helped explain previous conflicting results, whereby the isolated NS1 proteins from certain naturally occurring and laboratory-adapted virus strains appeared to lack the ability to bind/inhibit CPSF30.^[74,103,110] An additional mechanism that NS1 may utilize to limit gene expression is to block cellular mRNA export from the nucleus,^{[106,111,112]} possibly by binding and inhibiting components of the nuclear mRNA export machinery (Figure 1).^[113] The relative contributions to IFN inhibition of NS1 specifically targeting the RIG-I pathway versus its ability to limit general gene expression have not yet been addressed, and may vary between different virus strains. Furthermore, blocking general gene expression may limit other non-IFN-related pathways that also benefit virus replication. For example, very recent studies have specifically implicated the 1918 pandemic influenza A virus NS1 protein in blocking expression of genes related to lipid metabolism,^[114] although the functional consequences of this have yet to be established.

The heterotrimeric viral polymerase complex, polymerase subunit basic (PB)1–PB2–polymerase subunit acidic (PA), mediates a cellular mRNA cap-snatching activity that is essential for priming transcription of viral mRNAs.^[115] Inevitably, this will reduce the levels of capped host mRNAs that are translated into functional proteins and may, therefore, constitute an additional mechanism by which influenza viruses attenuate host cell gene expression, including that of IFN-β (Figure 1).^[56] Mechanistically, the PB1 protein indirectly participates in cap-snatching activity, in that once bound to the viral template it activates the cap-binding activity of the PB2 subunit.^[116,117] The PA subunit has recently been shown to encode the endonuclease activity that removes the cap from the host mRNA.^[118,119] There may be some interplay between the ability of the viral polymerase to shut down host-cell protein synthesis and the ability of NS1 to limit IFN induction by binding CPSF30, as all these virus and host components have recently been detected in the same complexes.^[120]

Influenza Viruses Limit PAMP Availability

As briefly discussed above, the byproducts of virus replication can be RNA PAMPs that activate the host innate IFN response. Although clearly also required for basic replication, the encapsidation of viral RNA into RNPs may be considered an additional mechanism by which influenza viruses limit the access of RIG-I to certain PAMPs. In particular, RNA encapsidation by nucleocapsid protein (NP) may limit the formation of dsRNA PAMPs by preventing annealing of negative- and positive-sense viral RNAs. Furthermore, given that PAMP sensors for RNA viruses have only been identified in the cytoplasm,^[13] the nuclear replication strategy of influenza viruses probably reduces the likelihood of PAMP detection (Figure 1).

Influenza Viruses Limit the Antiviral Effects of IFN

Several studies suggest that in addition to limiting IFN production, influenza viruses are also capable of downregulating signaling from type I and type II IFN receptors. First, influenza viruses have been reported to block signaling from the IFN-γ receptor by reducing the phosphorylation of STAT1 and its subsequent nuclear accumulation.^[121] In addition, influenza virus infection induces the expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit signaling from the IFN-α/βR (Figure 2).^[122,123] It is currently unclear whether influenza viruses actively suppress these IFN signaling pathways, or if these responses are part of the normal regulatory feedback mechanisms of the cell. Furthermore, given that the antiviral effects of IFNs require transcriptional upregulation of target genes, the ability of influenza viruses to efficiently shut down host cell protein synthesis (either via NS1-mediated^[104] or cap-snatching mechanisms^[115]) is also likely to dampen the IFN response. The apparent capacity of some influenza viruses to block IFN signaling at multiple levels may mean that these viruses are particularly efficient at preventing the establishment of an antiviral state within cells.^[103] In this regard, it is noteworthy that a naturally occurring polymorphism (D92E) has been reported in NS1 that enhances its ability to limit the IFN-induced antiviral state, and this amino acid change is associated with influenza A virus virulence.^[124]

Influenza Viruses Limit the Effects of IFN-inducible Antiviral Effectors

Surprisingly little is known about the roles of specific antiviral effector proteins in downregulating influenza virus replication. Remarkably, for those that have been partially characterized (such as PKR, OAS, ISG15 and MxA), it is clear that influenza viruses have evolved strategies to modulate their own sensitivity to effector-mediated inhibition. Influenza A viruses use two mechanisms to counteract the powerful antiviral activity of PKR.^[125] First, influenza A virus infection upregulates the activity of the cellular inhibitor of PKR, p58^IPK (Figure 2).^[126] The mechanism by which this upregulation occurs is not entirely clear, although there is no increase in the total amount of p58^IPK protein.^[127] Rather, evidence suggests that influenza A virus infection causes the dissociation of p58^IPK from its natural cellular inhibitor, heat shock protein 40.^[128,129] The influenza A virus NS1 protein has also been reported to bind directly to PKR in order to inhibit the conformational changes that regulate its activity.^[130] Similarly, the influenza B virus NS1 protein is able to limit PKR activity, but this seems to be mediated by a distinct mechanism involving a dsRNA bridge between NS1 and PKR.^[131,132] A key additional function of dsRNA binding by the influenza A virus NS1 protein appears to be the inhibition of OAS, possibly by sequestration of viral dsRNA.^[130] Whether the influenza B virus NS1 protein also counters OAS by dsRNA sequestration is unknown.

One apparently unique function of the influenza B virus NS1 protein is its ability to bind ISG15 and subsequently inhibit the IFN-stimulated conjugation of ISG15 to cellular proteins.^[133] This property is not shared by the influenza A virus NS1 protein. The mechanism by which influenza B virus NS1 inhibits ISG15 conjugation is far from fully established, but is likely to rely on the disruption of key interactions between ISG15 and the cellular E1/E2/E3 activation and ligation machinery.^[133,134]

It is probable that most (if not all) antiviral effectors activated by influenza virus infections are to some degree circumvented by a virus strategy. The extent to which a particular virus is able to achieve this can vary from one strain to another, a factor that can be owing to species-specific adaptations and which may ultimately contribute towards virulence. For example, the highly pathogenic 1918 influenza A virus RNP complex appears completely insensitive to the antiviral effects of human MxA, whilst more contemporary nonpathogenic human viruses are mildly sensitive to MxA.^[135] Intriguingly, many avian influenza A viruses seem highly susceptible to the antiviral actions of human MxA.^[135] A similar strategy has been described whereby a highly virulent influenza A virus strain can effectively 'out-run' the antiviral restrictions mediated by Mx proteins, apparently by simply replicating faster than other low-pathogenic, Mx-sensitive virus strains.^[136,137]

Influenza Viruses Modulate Cell Death

In cells and tissues in which influenza viruses replicate, the induction of apoptotic cell death may be considered an antiviral mechanism by which the host attempts to limit viral spread. As such, influenza viruses may indirectly evade the antiviral effects of apoptosis by replicating at faster speeds (Figure 2).^[138] There have also been reports that the influenza A virus NS1 protein can inhibit apoptosis in infected cells.^[139–141] This can be attributed, at least in part, to its ability to suppress both IFN induction and the antiviral effects of IFN.^[140] Furthermore, evidence suggests that activation of PI3K signaling by NS1 may directly contribute to suppression of host cell apoptosis (Figure 2).^{[139,141–143]} The NS1 protein of influenza B viruses does not bind or activate PI3K,^[144] and the role of this NS1 protein in limiting apoptosis during infection has yet to be firmly established.

Rather than blocking apoptosis, several studies have concluded that influenza viruses actually enhance cell death for a virus-supportive function. It is conceivable that by killing infected cells, virus release is enhanced. In addition, stimulating apoptosis may reduce cell-mediated cytotoxic responses, as infected cells are cleared by phagocytosis. A recent study has proposed that influenza A viruses target and destroy natural killer cells, thereby reducing the pool of innate immune cells that normally help to clear virus infections.^[145] The mechanism of this immune cell killing is unknown, but could be due in part to expression of the viral PB1 frame 2 (PB1-F2) protein. PB1-F2 is a small 87-amino acid protein encoded by an alternate (+1) reading frame within the PB1 gene.^[146] PB1-F2 localizes to mitochondrial membranes,^[146–148] and its expression induces the formation of nonspecific pores within membranes.^[149] PB1-F2 interacts with the mitochondrial apoptotic mediators adenine nucleotide translocator 3 and voltage-dependent anion channel 1,^[150] thereby sensitizing cells to apoptotic cell death.^{[146,147,149–151]} The proapoptotic effect of PB1-F2 is most pronounced in 'immune' monocyte/macrophage cells,^[146] and data from both reassortant and mutant viruses indicate that PB1-F2 contributes significantly towards viral pathogenesis.^[152–154] Mechanistically, it has therefore been suggested that the proapoptotic function of PB1-F2 serves to limit efficient immune-cell-mediated virus clearance in vivo.^[152] It should be noted that other influenza virus proteins have been linked to induction of apoptosis (e.g., NA^[155,156] and matrix protein 1 [M1]^[157]), but the biological reasons for these functions remain unclear. Interestingly, a connection between influenza A virus-induced apoptosis and perturbation of autophagosome maturation by the viral M2 ion channel has recently been established.^[158] However, how regulation of autophagosomes contributes to influenza A virus replication and virulence remains to be determined.^[158,159]

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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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