Interferon-inducible antiviral effectors

doi:10.1038/nri2314

Nat Rev Immunol. Author manuscript; available in PMC 2009 January 1.

Published in final edited form as:

Nat Rev Immunol. 2008 July; 8(7): 559–568.

doi: 10.1038/nri2314

PMCID: PMC2522268

NIHMSID: NIHMS56475

Interferon-inducible antiviral effectors

Anthony J. Sadler and Bryan R. G. Williams

Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia

Correspondence to B.R.G.W. e-mail: bryan.williams/at/med.monash.edu.au

The publisher's final edited version of this article is available at Nat Rev Immunol

See other articles in PMC that cite the published article.

Preface

Since the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and their antiviral activities. Gene targeting studies have distinguished four effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, the 2′-5′ oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R pathway and the ISG15 ubiquitin-like pathway. These effector pathways individually block viral transcription, degrade viral RNA, inhibit translation, and modify protein function to control all steps of viral replication. Ongoing research continues to expose additional activities for the effector proteins and has revealed unanticipated functions of the antiviral response.

Introduction

Interferon (IFN) was discovered more than 50 years ago as an agent that inhibited the replication of influenza virus¹. The IFN family of cytokines are now recognized as key components of the innate immune response and the first line of defence against virus infection. Accordingly, IFNs are currently used therapeutically, with the most noteworthy example, to combat Hepatitis C viral (HCV) infection, but also against a range of other disorders, including numerous malignancies and multiple sclerosis (recently reviewed²).

Three classes of IFN have been identified, designated types I to III, which are classified according to the receptor complex they signal through (Figure 1). Type II IFN consists of the single IFNγ gene product that binds the IFNGR receptor complex. IFNγ mediates broad immune responses to pathogens other than viruses. The more recently described type III IFNs include three IFNλ gene products that signal via the combined IFNLR1 and interleukin-10 receptor 2 (IL-10R2) receptors. To date little is known about the type III IFNs, although they are known to regulate the antiviral response and have been proposed to be the ancestral type I IFNs³. Type I IFNs, which in humans comprise 13 IFNα subtypes, IFNβ, IFNκ, IFNε, IFNo, IFNτ and IFNδ, engage the ubiquitously expressed IFNα receptor (IFNAR) complex that is composed of the two components, IFNAR1 and IFNAR2. The function of type I IFNs is well characterized and they are known to be essential for mounting a robust host response against viral infection. Accordingly, IFNAR-deficient mice have increased susceptibility to numerous viruses but maintain resistance to other microbial pathogens, such as Listeria monocytogenes⁴^,⁵. Similarly, humans with genetic defects in components of IFN signalling (STAT1, TYK2 or UNC93B) die of viral disease, with the defect in type I IFN (rather than IFNγ) signalling having the more significant role⁶^-⁹.

Figure 1

IFN-signalling

Binding of type I IFNs to IFNAR, with ensuing signal transduction, leads to the induction of more than 300 IFN-stimulated genes (ISGs)¹⁰. However, relatively few of these ISGs have been directly implicated in instigating the antiviral state. Instead, many of the gene products encode pattern-recognition receptors (PRRs) that detect viral molecules and modulate signalling pathways, or transcription factors that form an amplification loop resulting in increased IFN production and protection from virus spread to limit disease. ISGs that encode promising candidates with direct antiviral activity include proteins that catalyse cytoskeletal remodelling, that induce apoptosis and that regulate post-transcriptional events, such as splicing, mRNA editing, RNA degradation and the multiple steps of protein translation, as well as subsequent post-translational modification. Indeed, several such proteins, ISG15, the GTPase Mx1, ribonuclease L (RNaseL) and protein kinase R (PKR) have been validated as antiviral effectors in studies of gene knockout mice. Mice with mutations or deficiency in key steps in the pathways triggered by these proteins have increased susceptibility to virus infection.

In this Review, we summarize our current understanding of the role of these four antiviral proteins. However, it bears mentioning that this isn't the complete antiviral repertoire. Additional ISGs with likely significant roles in antiviral activities are the deaminases ADAR1 and APOBECS, the exonuclease ISG20, members of the tripartite motif proteins such as PML, the putative S-adenosyl-L-methione Viperin, and the highly induced translation regulators IFIT1 and IFIT2. All have been reported to function as antiviral proteins but await better description for their relative importance using the appropriate knockout models. In addition, responses elicited by IFN-induced microRNA are emerging as regulators of viral infection¹¹.

ISG15, the Mx proteins, the 2′-5′ oligoadenylate synthetase (OAS)-directed RNaseL pathway and PKR represent a gradient of IFN responsiveness: ISG15 is one of the most highly induced ISGs and when coupled to protein substrates modulates pleiotropic cellular activities; Mx proteins are also highly induced by IFN then self-assemble into oligomers that are constitutively active; OAS proteins are expressed at low levels, then considerably induced by type I IFNs that are subsequently activated by viral RNA; and PKR is constitutively expressed as an inactive kinase that is activated by viral double-stranded RNA (dsRNA), then further induced by IFN. This variable responsiveness to IFN underlies the function of each protein as solely an IFN effector, or as determinants of innate immunity and PRRs to enhance the IFN response.

ISG15

One of the most prominent ISGs induced during viral infection and the ensuing IFN response is the 17 kDa ISG15. Although the gene was cloned over 20 years ago¹², an antiviral function for ISG15 has only recently been established, and considerable work is still required to detail all of its actions and to resolve contradictory findings.

ISG15 was identified soon after the landmark discovery of ubiquitin, and was immediately recognised as a ubiquitin homologue (Figure 2)¹³. Protein ubiquitylation regulates many aspects of immunity, including intracellular signal transduction, for instance via activation of NF-κB, as well as acquired immune functions, such as initiating tolerance (reviewed by Liu et al.¹⁴). Given the importance of ubiquitylation in the immune response, it is perhaps not surprising that there is a tailored IFN-regulated ubiquitin-like protein response. This response, as mediated by ISGs, has been coined ISGylation.

Figure 2

Domain structure of antiviral proteins

ISG15 is expressed as a 165 amino acid precursor that is subsequently processed to expose the C-terminal sequence LRLRGG. The equivalent diglycine residues within this motif on ubiquitin are adenlylated and conjugated by a thiolester bond to sequential cystine residues on the E1-activating, E2-carrier and E3-ligase enzymes, before being transferred to lysine residues on protein substrates. As the ubiquitin E1 enzyme (UBE1) is unable to form a thiolester bond with ISG15, ISGylation was initially thought to require a parallel and distinct pathway¹⁵. However, as the counterpart enzymes that catalyse ISGylation are identified, it is becoming apparent that there is direct interplay between these two pathways. The enzyme UBE1L (ubiquitin-activating enzyme E1-like) was shown to be the specific ISG15-activating enzyme¹⁶. Challenging this specificity, two E2 ubiquitin carrier enzymes, UBCH6 (also abbreviated UBE2E1) and UBCH8 (also abbreviated UBE2L6) were shown to also serve as ISG15 carriers¹⁷^,¹⁸. Ablation of UBCH8 by RNAi suggested this as the principal ISG15 E2 carrier in HeLa cells¹⁹. Finally, two E3 ubiquitin ligases, HERC5 (HECT domain and RLD 5) and TRIM25 (Tripartite Motif Protein 25), have been identified to also conjugate ISG15 to protein substrates, via their respective HECT (Homologous to the E6-Ap Carboxyl Terminus) or RING (Really Interesting New Gene) domains²⁰^,²¹. Appropriately, all enzymes identified in the ISGylation pathway are coordinately induced by IFN (Figure 3). As for ubiquitylation, ISGylation is reversible and a number of enzymes that catalyse the hydrolysis of ISG15 (termed deISGylation) have been identified, including the ubiquitin-specific protease-18 (USP18, also abbreviated UBP43), USP2, USP5, USP13 and USP14²²^,²³.

Figure 3

Mechanism of action of ISG15

At least 158 putative ISG15 target proteins have been identified to date²⁴^-²⁶. Many of these substrates are important in the IFN response, including the signalling components JAK1 and STAT1, PRRs such as RIG-I (retinoic-acid-inducible gene I), and the antiviral effector proteins MxA, PKR and RNaseL²⁴. Unlike ubiquitylation, ISGylation does not drive protein degradation (regulated by K48-linked ubiquitin), but rather parallels ubiquitin's activating effects (mediated by K63-linkage). Accordingly, ISG15 has been reported to prevent virus-mediated degradation of the IFN regulatory factor 3 (IRF3), thereby enhancing induction of IFNβ²⁷. ISGylation has also been shown to modulate the function of enzymes. An instance of this is the increased affinity of the Eukaryotic Translation Initiation Factor 4E family member 4EHP for the 5′ cap structure of RNA²⁸. Conversely, conjugation of ISG15 to the Protein Phosphatase 1B (PPM1B) suppressed enzyme activity, thereby enhancing NF-κB signalling²⁹.

In addition to its intracellular role, ISG15 is secreted in large amounts and has been shown to act as a cytokine to modulate immune responses³⁰. The mechanism by which extracellular ISG15 functions is unresolved. Ubiquitin is also secreted from cells with immunomodulatory effects that are not understood³¹. However, extrinsic ubiquitylation has been claimed through analysis of surface proteins on spermatozoa during post-testicular maturation³². Conceivably, secreted ISG15 may also function in extrinsic ISGylation. This intriguing possibility could be tested by the treatment of Ube1l-/- mice with ISG15.

Crucial for designation as an antiviral protein, mice ablated for Isg15 have increased susceptibility to a number of viruses, including the influenza A and B viruses, Sindbis virus (SV), and both herpes simplex virus 1 (HSV-1) and murine γ–herpesvirus³³^,³⁴. Supporting this, chimeric SV expressing ISG15 are protected from the otherwise lethality of the wild-type viral infection in Ifnar1-/- mice. Compellingly, this rescue effect was dependent on the integrity of the conserved LRLRGG residues at the C-terminus of ISG15³⁵. Confounding these reports, a similar chimeric ISG15-SV did not rescue the same Ifnar1-/- mouse, as reported by another laboratory. However, the recombinant SV construct did provide modest protection in vitro³⁶. Ablation of the deISGylation enzyme, USP18, in mice increased resistance to virus infection, notably VSV. However, the expected reciprocal sensitivity to VSV is not apparent in either the Isg15-/- or Ube1l-/- mice³⁷. However, Isg15-/- mouse embryonic fibroblasts were more susceptible to VSV infection, although this sensitivity was lost after IFN treatment, suggesting circulating IFNs in vivo may obscure some viral resistance mechanisms²⁷. Other in vitro experiments support a role for ISG15 in mediating resistance to Ebola virus, via ISGylation of the E3 ubiquitin ligase NEDD4 (Neural Precursor Cell Expressed, Developmentally Downregulated-4), thereby preventing subsequent ubiquitylation³⁸. Similarly, HIV-1 Gag and Tsg101 ubiquitylation is inhibited by ISG15³⁹. Both NEDD and Gag/Tsg101 ubiquitylation mediate virion release from cells. Further corroboration of an antiviral role for ISG15 comes from the identification of viral proteins that have evolved to target different steps in ISGylation. The Nonstructural protein-1 (NS1) of the influenza B virus binds ISG15 to block ISGylation¹⁶^,⁴⁰. Finally, a number of viral proteases from SARS coronavirus, crimean-congo hemorrhagic fever virus, equine arteritis virus, porcine respiratory and reproductive syndrome virus, and SV have been identified to mediate deISGylation⁴¹^-⁴³.

As mentioned, one of the substrates modified by ISG15 are the Mx proteins²⁴.

Mx GTPases

IFNs induce the expression of several guanine-hydrolysing proteins. This class of protein is involved in scission to mediate vesicle budding, organogenesis and cytokinesis. There is evidence that four families within this protein class: the p47 guanylate-binding proteins (GBPs), the p65 GBPs, the very large inducible GTPases (VLIGs) and the Mx proteins, afford resistance to pathogens⁴⁴. However, of these only the Mx proteins have a well-characterized antiviral role and show a strict dependence on type I and III IFN for their expression⁴⁵.

The Mx family of GTPases, which comprises MxA and MxB in humans and Mx1 and Mx2 in mice, were first identified as antiviral proteins by the observation that the sensitivity of many inbred mouse strains to orthomyxomavirus was solely due to arbitrary mutations within the Mx locus on chromosome 16⁴⁶^-⁴⁸. This sensitivity could be rescued by restoration of Mx1 expression⁴⁹. Strikingly, constitutive expression of the human equivalent of mouse Mx1, MxA, in IFNAR-deficient mice confers full resistance to an otherwise fatal infection with Thogoto virus (THOV), LaCrosse Virus (LACV) and Semliki Forest virus (SFV)⁴⁹.

The two human Mx proteins are encoded on chromosome 21, in a region syngeneic to the Mx region on mouse chromosome 16⁵⁰^,⁵¹. The human proteins are cytoplasmic, as is the murine Mx2. However, the murine Mx1 is nuclear. This differential distribution is thought to allow each protein to target viruses that replicate in either cell compartment⁵². Only human MxA has demonstrated antiviral activity, and this is directed against both nuclear and cytosolic viruses.

Considerable evidence now shows that mouse Mx proteins confer viral resistance. Viruses that are susceptible to the activities of Mx proteins include orthomyxomaviruses, paramyxomaviruses, rhabdoviruses, togaviruses and bunyaviruses. Similarly, human MxA has been shown to inhibit all infectious genera of the Bunyaviridae family (orthobunyavirus, hantavirus, phlebovirus and dugbe virus)⁵³. Members of other virus families, such as the clinically significant Coxsackie virus (from the Picornaviridae) and Hepatitis B virus (HBV) (Hepadnaviridae) are also susceptible to MxA⁵⁴^,⁵⁵. In addition, genetic studies of human populations have shown that a polymorphism in MxA correlates with increased susceptibility to Hepatitis C virus (HCV)⁵⁶ and HBV⁵⁷, as well as to measles virus, with the latter associated with higher rates of subacute sclerosing panencephalitis⁵⁸. Appropriately, Mx proteins are expressed in cells of the periphery, for example in hepatocytes, endothelial cells and immune cells, including peripheral blood monocytes, plasmacytoid dendritic cells and myeloid cells⁵⁹.

The Mx proteins have a large (relative to many GTPases) N-terminal GTPase domain, a central interacting domain (CID) and a C-terminal leucine zipper (LZ) (Figure 2). Both the CID and LZ domains are required to recognize target viral structures. The main viral target appears to be viral nucleocapsid-like structures⁶⁰. By virtue of their location at the smooth endoplasmic reticulum, Mx proteins can effectively police exocytic events and mediate vesicle trafficking to trap essential viral components, and in so doing, they prevent viral replication at early time points (Figure 4)⁶¹. Also, both MxA and Mx1 associate with subunits of the influenza virus polymerase (PB2 and nucleocapsid protein) to block transcription⁶². This is a potent antiviral measure, which effectively prevents the generation of viral Mx escape mutants. As a result, few viral countermeasures against Mx proteins have been identified.

Figure 4

Mechanism of action of MxA

Most viral escape mechanisms that are described target IFN signalling: for example, highly virulent strains of influenza virus increase their replicative fitness to effectively out run the IFN response⁶³. More directly, the HBV precore/core protein has been reported to interact with the MXA promoter to prevent MXA gene expression⁶⁴. Also, West Nile virus (WNV) produces, what appear to be, decoy cytoplasmic membrane structures to hide crucial replication components⁶⁵.

In contrast to ISG15 and Mx, the OAS and RNaseL pathways and PKR are present ubiquitiously at constitutive levels but can be amplified by exposure to IFNs.

The OAS and RNaseL pathway

Initially identified as IFN-induced proteins that generate low-molecular weight inhibitors of cell-free protein synthesis, the OAS proteins are distinguished by their capacity to synthesize 2′, 5′-linked phosphodiester bonds to polymerize ATP into oligomers of adenosine⁶⁶^,⁶⁷. These unique 2′, 5′-oligomers specifically activate the latent form of RNaseL leading to RNA degradation⁶⁸. In this way, OAS in combination with RNaseL constitutes an antiviral RNA decay pathway. As the OAS proteins are constitutively expressed at low levels they can act as PRRs for the detection of viral dsRNA in the cytosol⁶⁵. RNA degraded by RNaseL is able to activate the other cytoplasmic PRRs RIG-I and MDA5 (melanoma differentiation-associated gene 5) resulting in IFN gene induction. This accounts for the observation that RNaseL-deficient cells show markedly decreased IFNβ production due to reduced signalling via these PRRs⁶⁹.

The four OAS genes identified in humans, termed OAS1, OAS2, OAS3 and OASL (OAS-like), have been mapped to chromosome 12 (chromosome 5 in mice) (reviewed by Hovanessian and Justesen⁷⁰). OAS1 has two spliced forms in humans (eight in mice) that produce two, 40 and 46 kDa, proteins that differ at their C-termini by 18 and 54 amino acids, respectively. OAS2 produces four alternatively spliced transcripts that encode two proteins of 69 and 71 kDa. OAS3 encodes a single transcript that produces a 100 kDa protein. These proteins have considerable homology to each other, with OAS1, OAS2 and OAS3 encoding one, two and three, respectively, ‘OAS’ domains (Figure 2). The most distinctive of the OAS proteins is OASL. Two OASL transcripts are expressed producing two proteins of 30 and 59 kDa. The higher molecular weight OASL contains a putative nucleolar localization signal (RKVKEKIRRTR) at its C-terminus that, probably, accounts for its unique (from the other OAS isoforms) distribution in the cell. The OASL protein also has an OAS domain, however, mutations at key residues disable the catalytic function of this human protein. Interestingly, one of the two mouse homologues retains its 2′, 5′-polymerase activity. In addition to the OAS domain, OASL has a unique 160 amino acid C-terminus that encodes a ubiquitin-like domain that is homologous to ISG15. Accordingly, OASL becomes conjugated (ISGylation) to cellular proteins following the treatment of cells with type I IFNs⁷¹.

There appears to be differential expression and induction of each form of the human OAS proteins⁷². Also, each of the three functional OAS proteins has unique biological functions. A tripeptide motif (CFK) within the OAS domains of OAS1 and OAS2 mediate oligomerization, so the catalytically active form of these enzymes is a tetramer and dimer, for OAS1 and OAS2, respectively⁷³. This tripeptide motif is not conserved in the OAS domains of OAS3 and OASL and therefore these proteins function as monomers. The polymerization of OAS monomers influences their processivity, with OAS3 synthesizing dimeric molecules of 2′, 5′-linked oligomers, whereas OAS1 and OAS2 are capable of synthesizing trimeric and tetrameric oligomers⁷⁴^,⁷⁵. The dimeric 2′, 5′-linked oligomers are not efficient activators of RNaseL⁷⁶ and, consequently, are thought to regulate alternative processes, with one report suggesting a role in gene expression by regulating DNA topoisomerase I⁷⁷.

The 2′, 5′-dependent RNaseL is expressed as an 80 kDa protein with two kinase-like domains (PUG and STYKc) and eight ankyrin repeats (Figure 2) (reviewed by Silverman⁷⁸). The enzyme is constitutively expressed as an inactive monomer. Autoinhibition of the enzyme is relieved upon binding of 2′, 5′-oligomers (generated by OAS proteins) to the ankyrin repeats, and subsequent homodimerization⁷⁹. The active dimeric enzyme then degrades ssRNA (Figure 5)⁸⁰^,⁸¹. Due to their perceived common action via RNaseL, the antiviral function of the OAS proteins has been investigated using RNaseL-deficient mice⁸². These mice show increased susceptibility to RNA viruses from the Picornaviridae, Reoviridae, Togaviridae, Paramyxoviridae, Orthomyxoviridae, Flaviviridae and Retroviridae families⁷⁸. An antiviral role for RNaseL against DNA viruses is less directly established, although as these viruses produce dsRNA replicative intermediates they can induce 2′, 5′-oligomers. However, ensuing activation of RNaseL is less commonly observed, presumably because of virally encoded inhibitory factors. A case in point is the E3L protein from Vaccinia virus (VV)⁸³. The direct importance of OAS proteins in the antiviral response in humans is highlighted by genetic studies showing that polymorphisms within a splice-acceptor site of the OAS1 gene (producing two isoforms of the enzyme with different activities) significantly correlate with the antiviral response to the yellow fever vaccine in immunization trials⁸⁴.

Figure 5

The OAS-RNaseL antiviral pathway

It has recently become apparent that the OAS proteins have additional antiviral functions that are independent of RNaseL activity. The precise mechanisms of RNaseL independence remain to be elucidated. Nevertheless, single nucleotide polymorphisms (SNPs) at a splice enhancer site in OASL have been correlated with susceptibility to WNV⁸⁵. Intriguingly, the capacity to accept GTP has suggested a potential role for OAS in RNA splicing, whereby the enzyme generates a 2′, 5′-phosphodiester bond between the G at the 5′ end of an intron, and the A of a 3′ splice signal in the splicing intermediate structure⁸⁶.

PKR

Similar to OAS, PKR (also known as EIF2αK2) was initially identified as a regulator of the antiviral response through studies of protein synthesis in cell-free lysates from IFN and dsRNA-treated cells⁶⁶^,⁸⁷. PKR belongs to a small family of protein kinases that respond to environmental stresses to regulate protein synthesis (the other members are EIF2αK1 (eukaryotic translation initiation factor 2α kinase 1; also known as HRI), EIF2αK3 (also known as PERK) and EIF2αK4 (also known as GCN2)). Members of this kinase family phosphorylate the translation initiation factor EIF2α at serine residue 51, resulting in sequestration of the limiting guanine nucleotide exchange factor EIF2β⁸⁸. This prevents recycling of GDP, halting translation, to allow the cell to reconfigure gene expression. Much of the antiviral and antiproliferative activities of PKR can be attributed to its phosphorylation of EIF2α. Moreover, structural determination of the complex of EIF2α and PKR argues against the existence of alternative substrates⁸⁹. However, there is extensive biological and biochemical evidence for alternative PKR targets, although the consequences of PKR phosphoregulation of other proteins have not been well characterized. As well as directly regulating proteins by phosphorylation, PKR evokes cellular responses by modulating cell-signalling pathways (discussed below).

PKR is constitutively expressed in all tissues at a basal level and is induced by type I and III IFNs⁹⁰. Under normal circumstances, PKR is maintained as an inactive monomer, through steric hindrance of the kinase domain by the N-terminus of the protein (Figure 2)⁹¹^,⁹². This repression is released by activating ligands, including viral RNAs, polyanionic molecules such as heparin⁹³ or ceramide⁹⁴, and protein activators⁹⁵ that elicit a conformational shift that permits the binding of ATP to the C-terminal kinase domain. The kinase domain consists of two lobes that separately regulate the interaction between protein monomers and the substrate. The active enzyme consists of a homodimer oriented in a parallel, back-to-back arrangement, with the active sites of the enzyme facing outwards⁸⁹. Dimerization elicits, and requires autophosphorylation at several key residues⁹⁶^-⁹⁸. Activation elicits phosphorylation of the EIF2α to halt translation (Figure 6).

Figure 6

Mechanism of action of PKR

Direct activation of PKR has been demonstrated with various RNAs by virtue of the two RNA-binding motifs (RBMs) in the N-terminal of PKR. All RBMs tested bind dsRNA independent of sequence, but recognize a specific higher ordered structure. Accordingly, PKR, like the OAS/RNaseL pathway, functions as a PRR. Although RBMs have been shown to bind just 16 base pairs of RNA, longer RNA moieties are required to engage both of the RBMs in PKR to activate the kinase⁹⁹. Consequently, dsRNA that is longer than 30 base pairs activates PKR most effectively. Alternatively, single-stranded RNAs of 47 bases that have limited ternary structure, activate the kinase if they carry 5′-triphosphates¹⁰⁰. As cellular RNA transcripts predominantly have 5′-monophosphates, this equips PKR to specifically target viral RNAs.

Other pathogen-associated molecules, such as lipopolysaccharide (LPS), which is a ligand of Toll-like receptor 4 (TLR4), can also activate PKR¹⁰¹, but this is likely indirect, via another protein. Accordingly, three protein activators of PKR have been identified. PKR interacts with the tumour-necrosis factor (TNF)-receptor-associated factor (TRAF) family of adaptor molecules that are integral to TLR signalling pathways¹⁰². The protein activator of PKR PACT responds to stress-inducing molecules, such as hydrogen peroxide, ceramide and cytokines (including IFNγ, IL-3 and TNF)⁹⁵. The consequences of activation of PKR by PACT in an antiviral context await further characterization of PACT-deficient mice. Finally, PKR can be activated by caspase-mediated (caspase-3, caspase-7 and caspase-8) cleavage of its inhibitory N-terminus, to generate a constitutively active, truncated, kinase domain¹⁰³^,¹⁰⁴.

The role of PKR has been investigated in mice using transgenic models. Deletion mutations have targeted both functional domains of the enzyme¹⁰⁵^,¹⁰⁶ and transgenic mice that are defective in PKR activity have been generated by expression of a trans-dominant negative mutant of human PKR that is defective in kinase activity (K296R)¹⁰⁷. Finally, transgenic mice overexpressing wild-type human PKR have also been produced¹⁰⁸. These transgenic mice have impaired antiviral responses with, for example, increased susceptibility to otherwise innocuous infections with VSV¹⁰⁹^,¹¹⁰, influenza virus and bunyawera virus¹¹¹. Experiments in PKR-deficient mouse embryonic fibroblasts show that PKR is involved in protection against infection with a number of RNA viruses, including HCV¹¹², Hepatitis D virus¹¹³, WNV¹¹⁴, HIV-1¹¹⁵, SV¹¹⁶, encephalomyocarditis virus (EMCV)¹¹⁷, and the Foot-and-mouth virus (FMDV)¹¹⁸, as well as some DNA viruses such as HSV-1¹¹⁹. As for MxA and OAS1, genetic analysis of human populations show that polymorphisms in PKR correlate with the outcome of HCV infection¹²⁰.

Additional immune functions of IFN-induced effectors

Studies of the ISG15, OAS/RNaseL and PKR pathways suggest additional functions for each of these proteins. The primary, activating enzyme in ISGylation, UBE1L, was recognized as being deleted in almost all small cell lung cancers, and so the gene product was speculated to be a tumour suppressor¹²¹^,¹²². Given the specificity of UBE1L in ISGylation, this presents an intriguing possible mechanism by which IFN could regulate proliferation.

RNaseL-deficient mice have an enlarged thymus and spleen due to suppressed apoptosis⁸². The significance of this ability of RNaseL to induce apoptosis has been exemplified through genetic analysis that identified a polymorphism (R462Q) that was associated with reduced enzyme activity and increased incidence of prostate cancer¹²³. Intriguingly, this SNP was subsequently associated with a putative oncolytic xenotropic murine leukaemia-related virus¹²⁴. Other nonviral functions of RNaseL are implied in experiments that show delayed skin-graft rejection in RNaseL-deficient mice¹²⁵. A polymorphism identified in OAS1 has also been associated with type I diabetes, which is consistent with a viral aetiology for this disease¹²⁶^,¹²⁷.

Given that PKR modulates several signalling pathways, a wider function than determined solely by regulation of translation would be expected. As mentioned, PKR is required for TLR4-mediated apoptosis in macrophages. Although PKR-mediated apoptosis is in part attributable to inhibition of translation through EIF2α phosphorylation, alternative signalling via IRF3 is also important¹⁰¹. PKR also promotes degradation of the inhibitor IκB, thereby activating the potent transcription factor NF-κB¹²⁸. Regulation of NF-κB accounts for the diminished NADPH oxidase 2 (NOS2) and IFNβ expression in PKR-deficient cells¹²⁹. Additionally, PKR was shown to be required for LPS-induced STAT inflammatory signalling¹³⁰. There is also a defect in IFN-induced phosphorylation of serine 727 in STAT1¹³¹, which is shown to be necessary for the basal expression of caspase-3. Accordingly, PKR-deficient fibroblasts are variably resistant to apoptosis induced by different stimuli, including dsRNA, LPS and TNF¹³². Conversely, over expression of PKR in NIH3T3 fibroblasts sensitises them to apoptosis¹⁰³. PKR may also influence the adaptive immune response by negatively regulating CD8⁺ T-cell function, as PKR-deficient mice have increased contact hypersensitivity responses and stimulus-dependent T-cell proliferation¹³³. PKR has also been implicated in IgE class switching in B cells. This points to a mechanism by which viral infection may induce IgE-mediated disorders, such as allergy and asthma¹³⁴^,¹³⁵. Surprisingly, these signalling events are not wholly mediated by direct phosphorylation by PKR, suggesting that the kinase acts as a scaffold to bridge signalling pathways from alternative PRRs¹³⁶. The mechanisms underlying these links to adaptive immunity are intriguing but remain to be explained.

Concluding remarks and future directions

The analyses of mice with targeted deletions in ISG15, Mx1, PKR and RNaseL have helped to elucidate specific roles for these enzymes in the response to virus. Further details of the mechanisms for each of these effectors are still to be elucidated. Less well characterized is the ISG15 pathway. Considerable work is still required to decipher the processes of ISGylation, to characterize protein substrates and to detail the consequence for the antiviral response. Also, the precise function of the Mx proteins remains uncertain. The contribution of alternative PKR substrates (beside EIF2α) to the immune response are poorly explored, and questions remain about specific roles for PKR in regulating inflammatory responses and whether this requires the kinase function or indicates a role as an adaptor protein. The precise roles of the different OAS proteins, especially relating to RNaseL-independent antiviral effects, are ill-defined. These alternative functions will be better addressed in vivo by the analysis of mice with more subtle targeted mutations, and in vitro by detailed biochemical analyses (for example, by identification of specific phosphorylation sites on substrates for PKR) and by more detailed structural investigation of the mechanism of activation of each protein.

Strategies used by viruses to escape these antiviral pathways are also informative and a large number of viral countermeasures to block ISG15, PKR, RNaseL and Mx have been reported (reviewed⁷⁸^,¹³⁷^,¹³⁸). It should be noted however, that many of the mechanisms attributed to virus-evolved avoidance of antiviral effectors and enhanced virulence have not been rigorously proven. An exception to this is the mechanism developed by the HSV-1 protein γ1ICP34.5 for targeting PKR. In this case, a virus that has been attenuated by deletion of ICP34.5 only replicates as efficiently as wild-type virus when it infects PKR-deficient mice. Restoration of virulence was shown to depend on ICP34.5 by using both host and viral mutants¹³⁹. Although this approach is not trivial to replicate, it remains the most valid method of defining effective viral avoidance of IFN activity.

Another important development currently underway is genetic studies that catalogue SNPs in each of these genetic loci within human populations and other animal species. Correlation of genetic polymorphisms in putative antiviral genes with susceptibility to virus, as well as incidence of other immune responses, is emerging as a powerful means to confirm gene function and measure their contribution to disease.

Finally, the most interesting developments in this field are likely to come from further insights into the broader role of each of these antiviral proteins, particularly in mediating adaptive immunity. While the mechanisms underlying these links to adaptive immunity remain to be explained, progress in this area has great potential to realize the goal of manipulating immunity to enhance resistance to pathogens and disease or, alternatively, diminish deleterious autoimmune responses. This will probably also require parallel advancement in our understanding of the specificities of type I and III IFN signalling.

Acknowledgments

Work in our laboratory is supported by grants from the US National Institutes of Health (P01 CA062220 and R01 AI034039) and the Australian National Health and Medical Research Council (436814).

Glossary


Pattern-recognition receptors	(PRRs). A host receptor that can sense pathogen-associated molecular patterns and initiate signalling cascades that lead to an innate immune response. These can be membrane bound (e.g. TLRs) or soluble cytoplasmic receptors (e.g. RIG-I, MDA5 and NLRs).

plasmacytoid dendritic cells	(pDCs). A unique type of dendritic cell with a morphology that resembles that of a plasmablast. These cells are also known as interferon (IFN)-producing cells because they are the main source of type I IFNs during viral infections.

suppressors of cytokine signalling	(SOCS). Intracellular proteins that are cytokine-induced negative regulators of cytokine signalling.

TNF-receptor associated factors	(TRAFs). A family of conserved scaffold proteins that link receptors of the TNF and Toll/IL-1 receptor family to various protein kinases (e.g. IRAK1). These proteins encode RING domains that, by association with E3 ubiquitin ligases, catalyze polyubiquitylation of proteins (e.g. IKKs to activate the transcription factors NF-κB) to transduce signals within the cell.

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