Danger, diversity and priming in innate antiviral immunity

Accepted Manuscript Title: Danger, Diversity and Priming in Innate Antiviral Immunity Author: Susan E. Collins Karen L. Mossman PII: DOI: Reference:

S1359-6101(14)00064-1 http://dx.doi.org/doi:10.1016/j.cytogfr.2014.07.002 CGFR 803

To appear in:

Cytokine & Growth Factor Reviews

Received date: Accepted date:

30-6-2014 3-7-2014

Please cite this article as: Collins SE, Mossman KL, Danger, Diversity and Priming in Innate Antiviral Immunity, Cytokine and Growth Factor Reviews (2014), http://dx.doi.org/10.1016/j.cytogfr.2014.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Danger, Diversity and Priming in Innate Antiviral Immunity

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Susan E. Collins and Karen L. Mossman*

Department of Pathology and Molecular Medicine, McMaster Immunology Research Center,

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Institute for Infectious Disease Research, McMaster University, Hamilton, Canada L8S 4K1

1280 Main Street West, MDCL 5026

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McMaster University

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*Corresponding author:

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Hamilton, Ontario, Canada L8S 4K1

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Phone: 1-905-525-9140 x23542 Email: [email protected]

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Abstract The prototypic response to viral infection involves the recognition of pathogen-associated

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molecular patterns (PAMPs) by pattern recognition receptors (PRRs), leading to the activation of transcription factors such as IRF3 and NFkB and production of type 1 IFN. While this response

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can lead to the induction of hundreds of IFN-stimulated genes (ISGs) and recruitment and

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activation of immune cells, such a comprehensive response is likely inappropriate for routine low level virus exposure. Moreover, viruses have evolved a plethora of immune evasion strategies to

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subvert antiviral signalling. There is emerging evidence that cells have developed very sensitive methods of detecting not only specific viral PAMPS, but also more general danger or stress

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signals associated with viral entry and replication. Such stress-induced cellular responses likely serve to prime cells to respond to further PAMP stimulation or allow for a rapid and localized

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intracellular response independent of IFN production and its potential immune sequelae. This review discusses diversity in innate antiviral players and pathways, the role of “danger” sensing,

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and how alternative pathways, such as the IFN-independent pathway, may serve to prime cells

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for further pathogen attack.

Keywords

Intrinsic and innate immunity; Interferon; Interferon-independent; Interferon regulatory factors; Danger; Priming

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Abbreviations

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FAST, fusion-associated small transmembrane; VLPs, virus-like particles

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1. Introduction

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The rapid response to viral infection is imperative for a cell, tissue and whole organism to gain the advantage in the interplay between host and virus. In the traditional model, viral

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infection is first detected via pattern recognition receptors (PRRs). At the cell surface, detection can be mediated following recognition of viral glycoproteins by surface receptors such as Toll-

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like receptors (TLRs) [1-3]. Following entry, viral nucleic acids act as pathogen associated

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molecular patterns (PAMPs) and are recognized via TLRs in the endosome or RIG-I-like receptors (RLRs) or DNA sensors in the cytoplasm. Whether the signal is glycoprotein, RNA or

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DNA, the detection of viral invasion ultimately leads to the activation of transcription factors, including interferon regulatory factor 3 (IRF3) and IRF7, which act cooperatively to produce

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small amounts of interferon beta (IFNβ). Secreted IFNβ then binds to its receptor on the infected cell and neighbouring cells and activates the JAK/STAT pathway to induce expression of a full

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array of interferon stimulated genes (ISGs) that collectively work to inhibit viral replication.

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This canonical pathway of innate antiviral immunity has been well reviewed [4, 5]. To ensure their survival, viruses have evolved diverse strategies to subvert every step of the antiviral response [4, 6, 7]. Accordingly, host cells have coevolved alternative signalling mechanisms that are independent of PRRs and the IFN response. Moreover, given that robust systemic immune responses can lead to destructive immune pathologies, it is intriguing to speculate that the host has similarly evolved more focused immune responses sufficient to control small scale or localized infections [8]. In this review, we will focus on diversity in the

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players and pathways in innate antiviral immunity, and the role of “danger” sensing, particularly

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in the context of routine, low-level pathogen exposure.

2. Mechanisms of Viral Detection: classic and alternative pattern recognition receptors

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2.1. Detection at the cell surface

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Surface expressed TLRs, including TLR1-TLR2, TLR2-TLR6 and TLR4, are considered the prototypic receptors for virus sensing and initiation of innate signal transduction pathways.

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However, these PRRs are either insufficient or even dispensable for initiation of antiviral signalling at the membrane during some infections. For example, we have shown a functional

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antiviral response in cells lacking TLR2- and TLR4-mediated signal transduction [9].

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Furthermore, TLR2 was shown to be dispensable for the antiviral response to Vaccinia virus delivered intra-dermally, although the adaptor MyD88 was required [10]. Alternative receptors,

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such as members of the lectin and scavenger receptor families, have also been identified as PRRs [11, 12]. Giannia and colleagues have shown that α5β3 integrin is involved in signalling the

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recognition of virus at the cells surface, either by co-ordinately working with TLR2 or through a TLR2-independent mechanism [13]. The TLR2-independent signalling occurs via a CARD9TRIF axis to activate IRF3 and IRF7. Tetherin, also known as CD317 or bone marrow stromal antigen 2, acts as an antiviral protein, by tethering virions at the cell membrane to prevent their release. However, it can also function as a cell surface detector of viral invasion [14]. Sensing of virus by tetherin activates the canonical NF
B signalling pathway, resulting in the expression of inflammatory cytokines. Thus, although the TLR pathway plays an important role in sensing viral invasion at the cell surface, there is increasing evidence that TLRs are not the sole

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mediators that initiate an antiviral response. There may be other cell surface receptors or complexes that act as first responders in the detection of a viral attack. The nature of such cell surface detectors may well be cell-type and stimulus-specific. Tetherin, for example, is

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constitutively expressed in a subset of cell types, such as B cells and plasmacytoid dendritic cells, but can be induced following stimulation with IFN [15]. Moreover, we and others have

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shown that an antiviral response to incoming enveloped virus particles requires not only virus

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to those mediated by cell surface recognition are required.

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binding to the cell surface, but also entry into the cell [16-18], suggesting that signals in addition

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2.2. Detection within the cell

Once a virus has entered the cell, its nucleic acid can be detected by intracellular PRRs. In

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the endosomal compartment, dsRNA is recognized by TLR3 that then signals through TRIF to activate NF
B and IRF3, while ssRNA is recognized by TLR7 and TLR8, which signal through

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the adaptor MyD88 [19]. Additionally, endosomal TLR9 has been shown to detect both CpG

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DNA and RNA:DNA hybrids [20]. Prototypic cytosolic sensors of viral dsRNA include the RLRs RIG-I and MDA5, which have different binding specificities, but ultimately both signal through the MAVS/TBK1/IRF3 axis. Recognition of RNA viruses has recently been reviewed [5, 21]. These RNA sensors are integral to the recognition of viral replication within the cell, even in the context of DNA virus infection, since replication of all viruses is thought to produce dsRNA intermediates [22]. In a manner similar to the RLRs, there are a number of cytosolic sensors of DNA, which converge on the ER-localized adaptor STING and signal through TBK1 and IRF3 [23]. Although the number of RNA sensors seems to be relatively restricted, there are

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an increasing number of proteins that have been identified as DNA sensors [24]. It is currently unclear why so many DNA sensors exist. These DNA sensors are predicted to respond to the genomes of incoming DNA virus particles, particularly when localized within the endosome or

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cytoplasm, as cellular DNA is normally restricted to the nucleus or mitochondria. Intriguingly, however, viral ssDNA or dsDNA can be sensed by IFI16 within the nucleus [25]. Recent

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“molecular ruler” to distinguish self-DNA from non-self DNA [26].

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evidence suggests that the ability of IFI16 to assemble into filaments on dsDNA creates a

While the recognition of intracellular viral RNA or DNA can act as a pathogen danger signal

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for the cell, there are some limitations. First, viruses have developed many strategies to evade detection by nucleic acid sensors, either by modifying or camouflaging their nucleic acids or by

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interfering with key components of the nucleic acid sensing machinery [27]. Further, by the time there is sufficient accumulation of nucleic acid in the cytoplasm to trigger a response, many

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viruses have efficiently initiated replication and are able to produce viral proteins for the purpose

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of circumventing the antiviral response [6, 7, 28]. Thus, to ensure the integrity of the host at the cellular and tissue levels, alternative sensing and protective mechanisms have evolved to respond

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to increasingly complex viral immune evasion strategies. Indeed, an emerging theme in the literature is the ability of cells to respond to “danger” signals associated with disruption of homeostasis upon pathogen invasion, independent of recognition of classic PAMPs.

3. Membrane Perturbation: signalling the “danger” of an incoming infection Although cell surface sensors play an important role in the recognition and restriction of viral entry and replication, given the importance of defending against pathogen attack, it stands

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to reason that cells possess diverse means of detecting viral invasion. One of the earliest signals warning of a viral infection is likely the physical disturbance associated with viral entry. During binding and entry, virus particles cause disruptions to the cellular membrane, cytoskeleton and

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endocytosis pathways [29, 30]. It has recently become clear that the cell has developed mechanisms to detect these global disturbances, even in the absence of traditional PAMPS and

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PRRs [8] (Figure 1).

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We, and others, have shown that cells can mount an antiviral response to lipo-protein complexes, such as enveloped viral particles, whether or not they are replication competent or

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contain nucleic acid [31, 32], by sensing perturbation of a cellular membrane. Detection of fusion as a danger signal confers an advantage to cells, as fusion at a cellular membrane is one of

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the earliest detectable steps in viral infection, and allows the cell to respond immediately, regardless of the invader. Moreover, initiating a response to an early generic process such as

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membrane perturbation precludes the need to encode multiple factors that respond specifically to

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defined PAMPs such as glycoproteins, ssRNA, dsRNA, 5’-pppRNA, ssDNA or dsDNA. As “fusion from without”, or polykaryocytosis, is relatively rare in biology in the absence of viral

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infection [33], it is tempting to speculate that such a process is recognized in most cell types as a “danger” signal.

Studies using the reovirus fusion-associated small transmembrane (FAST) p14 protein were able to show that membrane perturbation is the minimal required signal to generate an antiviral response in non-immune cells such as fibroblasts [32]. Further, this response generates the same restricted ISG profile as observed following the entry of a variety of enveloped virus particles, suggesting that it is the membrane perturbation upon viral entry that acts as the initial danger signal in response to viral infection. The response to membrane fusion requires IRF3, but is

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independent of IFN, much like the response to low level infection with either defective or replicating virus, where the threshold for activation of IRF3 was found to be much lower than that of NF
B [9, 34, 35]. In addition to direct activation of a subset of ISGs that are sufficient to

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limit virus replication following low-level virus exposure, the response to membrane perturbation may also prime the cell to respond to further stimulation by viral PAMPs, thus

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reducing the threshold of signal activation required to initiate the induction of IFN (Figure 2).

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While the response to membrane perturbation is IRF3 dependent, subsequent responses to viral dsRNA vary in their requirement for IRF3, with dependency decreasing with dsRNA length [36].

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Similarly, macrophages and epithelial cells recognize fusion of cellular and viral membranes to induce an IFN-dependent response that involves TBK1 and requires STING and IRF3 [31].

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Membrane perturbation was also shown to stimulate the PLC-PI(3)K pathway, leading to the

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release of calcium from the ER. This work was done with both virus-like particles (VLPs) and fusogenic liposomes. The level of ISGs induced by these stimuli was shown to correlate to the

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amount of fusion. Interestingly, stimulation by cell-virus membrane fusion enhanced the responsiveness of the cells to CpG and ssRNA, TLR9 and TLR7 ligands, respectively,

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reinforcing the hypothesis that membrane perturbation lowers the threshold of activation of conventional PRRs within the cell. Several studies have shown that additional physical changes induced in cells following viral fusion can also act as danger signals. Rearrangement of the cellular cytoskeleton, which is integral to the entry process for many viruses, can trigger protective responses in the cell. These responses involve detection of cytoskeletal rearrangements by kinases such as focal adhesion kinase (FAK) [37]. FAK has been shown to interact with the adaptor MAVS and participate in RLR signalling in an unknown mechanism that is independent of its kinase activity, but leads to

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the activation of both NF
B and type I IFN signalling pathways. FAK may act as a scaffold protein, facilitating the interaction of multiple signalling components. Focal adhesions are well situated at the membrane to detect incoming pathogens. Their connection to the actin

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cytoskeleton may allow the transmission of “pathogen invasion” signals to intracellular

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components such as mitochondrial-localized MAVS [38].

4. Other physical signals that indicate danger and induce innate immune signaling

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Non-viral entities such as silica, alum and diesel exhaust particles are also capable of inducing responses in cells and have been implicated in the generation of an inflammatory signal via

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activation of the NLRP3 inflammasome [39]. This observation supports the hypothesis that cells are capable of responding to diverse stimuli, even in the absence of classic PAMPs. The

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generation of an innate immune response to generic particulate matter may explain their efficacy as vaccine adjuvants, as an effective innate response has been shown to lead to a more efficient

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adaptive immune response [40]. Indeed, nanoparticles are currently being designed to mimic

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biophysical and biochemical cues of pathogens as vaccine delivery systems to stimulate a productive immune response without the destructive, pathogen-associated cytotoxicity [41]. Defects in homeostatic DNA repair pathways can also influence the innate immune response. Trex1 is a 3’ to 5’ repair exonuclease whose deficiency has been implicated in the pathogenesis of autoimmunity, including systemic lupus erythematosus, which displays a prominent IFN signature [42]. Accordingly, cells deficient in Trex1 have increased basal levels of ISGs [43]. The increase in basal ISG levels may be due to an accumulation of endogenous DNA in the cytoplasm, as the response is dependent on the DNA sensor cGAS [44]. Indeed, ER-associated

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Trex1 serves as a negative regulator of cytosolic viral DNA innate immune sensing [45]. Alternately, increased ISG levels may be attributed to an observed defect in lysosomal processing; Trex1 is a regulator of lysosomal biogenesis, and dysregulation of lysosomes can

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elicit a host innate immune response [43]. Moreover, Trex1 mutations in both the enzymatic and ER-localization domains have been linked with distinct human disease phenotypes, suggesting

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that Trex1 may have a function independent of its nuclease activity [46]. As with virus particles,

cell to prime the antiviral response to further danger signals.

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the IFN-independent response observed in the context of Trex1 deficiency may be a way for the

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In another study, using a siRNA library screening approach, components of the pyrimidine synthesis pathway were identified as mediators of the antiviral pathway [47]. When these

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components were knocked down, the resulting pyrimidine deprivation led to an increase in the expression of ISGs, in an IRF1-dependent manner. Similar results were seen when purine

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synthesis was inhibited with mycophenolic acid, leading to an IRF1-dependent increase in ISG

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expression [48]. Supplementing cells with exogenous purines or pyrimidines is not sufficient to reverse these antiviral effects, suggesting that it is the cellular stress of purine or pyrimidine

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imbalance that leads to induction of an antiviral response. ER stress has also been shown to activate the IRF3 pathway, in the absence of PRR stimulation, with different types of ER stress activating IRF3 via two distinct mechanisms [49]. ER stress that mobilizes calcium was shown to require both TBK1 and STING, suggesting that ER stress alone is using the same antiviral pathway induced by sensing of DNA viruses. However, the mobilization of calcium alone by ionomycin, in the absence of ER stress, was insufficient to induce the response, suggesting that there is some other component of the ER stress-induced unfolded protein response (UPR) that is required. Interestingly, other forms of

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ER stress were shown to activate IRF3 in an ATF6-dependent, but TBK-1- and STINGindependent manner. Whichever pathway was used, IRF3 activation was insufficient to induce expression of IFNβ, but rather acted synergistically with PAMPs such as LPS to elicit IFNβ

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induction. Similarly, ER stress has been demonstrated to synergize with both dsRNA and LPS to induce much higher levels of IFNβ [50]. This synergy is mediated by binding of the

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transcription factor XBP-1, which is associated with the ER stress-induced UPR, to an enhancer

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element upstream of the IFNβ promoter [51].

These findings again suggest that stress-induced cellular responses likely serve to prime

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cells to respond to further PAMP stimulation. Together, these results suggest that the cell has developed very sensitive methods of detecting not only specific viral PAMPS, but also more

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general danger or stress signals. These responses are seen in the context of physical disruptions such as membrane fusion and cytoskeletal rearrangement. They are also induced in the context

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of cell stress in the case of disruptions to the lysosomal processing pathway or nutrient

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deprivation observed by disruption of the pyrimidine or purine synthesis pathways. This sequential activation of the antiviral response offers substantial benefits to the affected cell. It

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allows the cell to augment its defences in the context of disruptions to either the physical or metabolic state of the cell, which may be innocuous in nature, or may instead represent a real threat in the form of invading pathogen.

5. The innate host response team: the usual suspects functioning in unusual ways 5.1. The prototypic signal transduction response

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Upon stimulation of non-immune cells by viral infection, IRF3 is activated by TBK1mediated phosphorylation, leading to its dimerization and nuclear translocation. In conjunction with transcription factors NFkB and AP-1, IRF3 assembles into an enhanceosome that recruits

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transcriptional machinery to the IFNβ promoter region [52]. IFNβ that is produced is released from the cell and binds to the IFNα/β receptor (IFNAR) in an autocrine and paracrine fashion,

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activating the JAK/STAT pathway and leading to the formation of the ISGF3 complex, which

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subsequently mediates transcription of genes containing an IFN-stimulated response element (ISRE) [53]. Among the products of these genes is IRF7. Once IRF7 has been produced and

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becomes activated, it can bind to the IFNα promoter, leading to a full IFN response and the subsequent induction of a comprehensive array of hundreds of ISGs [54, 55]. Recently, ELF4

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has been characterized as a cooperative binding partner that can increase the binding efficiency of either IRF3 or IRF7 to promoters [56]. The signalling in immune cells is somewhat different,

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as IRF7 is constitutively expressed at higher levels. This allows immune cells to proceed

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directly to IFNα induction in a much more rapid manner [57]. The importance of IRF3 and IRF7 in IFN production and the resulting antiviral response has

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been well established, leading to their designation as “master regulators” of innate antiviral immunity [58, 59]. Recently, however, there have been a number of studies demonstrating that under some conditions, in both immune and non-immune cells, either an incomplete enhanceosome or alternate IRFs can participate in the induction of an antiviral response.

5.2. Alternative signalling players and pathways

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We have shown that the response to low-level stimulation of fibroblasts with either virus particles or lipo-protein complexes occurs in the absence of IFN production [32, 34]. Instead, a small subset of ISGs is induced directly by IRF3, leading to an antiviral state. The ISGs induced

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are predominantly intracellular factors that function to block essential components of the virus life cycle, such as mRNA translation, or factors that enhance intrinsic and innate host responses.

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The lack of IFN production stems from a lower threshold for activation of IRF3 than NF
B;

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indeed, by increasing the level of stimulation with virus particles or lipo-protein complexes, IFN production ensues [9]. We have shown that IRF3 is essential to the IFN-independent response to

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membrane perturbation in fibroblasts, while IRF7 is dispensable [32, 34]. Given that membrane perturbation is independent of associated nucleic acids, it is not surprising that components of the

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TLR and RLR pathways are dispensable for this response [9, 60]. How membrane perturbation events trigger IRF3 activation is currently unclear. Indeed, activation through alternative

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pathways or in response to danger sensing likely accounts for the inability to identify a specific

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modification or feature that is indicative of IRF3 activation [61]. IFN-independent induction of ISGs has also been observed in response to stimuli that

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activate classic TLR and RLR pathways. Recently, RIG-I was shown to be induced in plasmacytoid dendritic cells following stimulation by TLR ligands in an IFN-independent fashion [62]. In the context of HCV infection of human hepatocytes, IRF3 and NF
B are directly recruited to the CXCL10 promoter following recognition of HCV by RIG-I or TLR3 [63]. Moreover, a study using human myeloid, epithelial and fibroblast cells showed that treatment of the cells with 5’ppp RNA prevents Dengue and Chikungunya virus replication, even when the cells are treated 4-8 hours after viral infection [64]. This response requires RIG-I, MAVS and IRF3, but is independent of IRF1, IRF7 and IFN. In contrast, the response to the

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synthetic dsRNA mimetic, polyIC, is IFN-dependent and mediated by TLR3 and MDA5. These findings suggest that cells are able to distinguish features of dsRNA and mobilize alternative antiviral signalling pathways. Direct binding of IRF3 to the promoter of a subset of ISGs, alone

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or in combination with other factors such as NF
B, allows for a more localized antiviral response that is independent of the sequelae associated with IFN signalling, and has likely

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evolved to protect the host against a low level or highly localized threat, with minimal

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engagement of the immune response [9].

Not surprisingly, viruses have evolved a myriad of strategies to render their environment

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IRF3 deficient [7, 65]. Accordingly, cells have co-evolved to elicit an antiviral response in the absence of IRF3. Fibroblasts lacking IRF3 respond to dsRNA, in a length-dependent manner.

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Responses to long dsRNA, indicative of viral genomes [36, 66], were found to be independent of IRF3 expression, whereas responses to shorter dsRNA molecules were entirely reliant on IRF3

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for generation of an antiviral response [36]. Similarly, many viruses can elicit IFN responses in

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the absence of IRF3. While Sin Nombre virus induces ISGs in the absence of IRF3, TLRs and RLRs [67], other viruses usurp components of the IRF, TLR and RLR pathways to mediate non-

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canonical signalling.

In murine bone marrow derived dendritic cells, a restriction of Measles virus replication was observed in the absence of both IRF3 and IRF7 [68]. These cells were still able to produce IFNβ, potentially by NF
B binding to the IFNβ promoter, although involvement of IRF1 or IRF5 was not ruled out. This pathway was specific to murine DCs and related only to the primary infection, as the surrounding cells were protected via released IFN. Similarly, the antiviral response to West Nile Virus has been shown to use alternative transcription factors, in the absence of IRF3 and IRF7 [69, 70]. In myeloid DCs, there is an IRF5 and MAVS dependent

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induction of IFNβ [69]. Unlike IRF3 or IRF1, however, IRF5 is not able to directly induce ISG expression. In macrophages, an antiviral response was observed that was independent of IRF3, IRF5 and IRF7, demonstrating that the transcription factors used in antiviral signalling may be

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both stimulus and cell-type specific. Mice that are deficient in IRF7 are able to restrict viral infection via NF
B [71]. This observation is consistent with previous reports that NF
B is

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capable of inducing a small subset of ISGs directly in cultured cells, in the absence of IFN

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signalling [72].

In murine embryonic fibroblasts (MEFs) and hepatocytes, an IRF1-dependent response was

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observed following HCV infection, in the absence of IFN production [73]. In MEFs, the response also required IRF5 and IRF7, whereas in hepatocytes the IRF1-dependent response was

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MAVS-dependent. IRF1 has previously been shown to bind directly to the promoters of IFNβ and other ISGs [74]. For example, IRF1 can directly induce the expression of the ISG viperin, in

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the absence of IFN, to control VSV replication [75]. This response was proposed to be a

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redundant mechanism that is used by the host cell when the virus has circumvented IFN production. As the same response was not observed in cells infected with NDV, this IRF1-

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dependent response may be virus-specific. IRF1 has also been shown to play an important role in the restriction of VSV replication in the neuronal setting [76]. In this model, IFN is an immediate early response factor, while IRF1 plays a non-redundant role in viral control during the late phase of infection. Thus IFN-independent pathways may have a non-redundant role to play by providing antiviral protection that is kinetically separated from the IFN response. In addition to the kinetic separation of the different arms of the antiviral response, RLR signalling can make use of physical separation to achieve different responses. MAVS, which was initially described as a mitochondrial protein, can also localize to peroxisomes [77].

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Peroxisome-localized MAVS signals through IRF1, in an IFN-independent manner, to generate an early antiviral response. In contrast, signalling through mitochondrial-localized MAVS is IFN-dependent and occurs with later kinetics.

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Finally, there is evidence that cells have evolved mechanisms to utilize components of the IFN production pathway to induce responses that are independent of both IFN and ISGs.

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Primary fibroblasts respond to dsRNA by inducing the expression of nitric oxide synthase in a

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manner that is IRF1 and NF
B dependent. This is an early response (2-5hr) that confers protection against subsequent infection by DNA viruses [78]. Of interest, although production of

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nitric oxide is independent of the IFN response status of the cell and is responsible for providing full antiviral protection in the absence of IFN and IRF3 signalling, it contributes significantly to

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host protection under conditions of a full IFN response.

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Of particular interest are observations of antiviral responses being elicited by factors outside of the “usual suspects” list. Recently, a role for STAT6 in antiviral activity was described [79].

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Detection of viral nucleic acids via MAVS (for VSV) or STING (for HSV) activates STAT6 in the absence of IFN production. STAT6 functions as a transcription factor to bind to promoters

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and induce expression of a subset of genes, leading to an antiviral response. STING and TBK1 are required for the response to both RNA and DNA viruses, with an additional requirement for MAVS in the detection of the RNA viruses [79]. The activation of STAT6 by virus occurs by a mechanism that is entirely distinct from its prototypic activation by IL-4 or IL-13. Virus induced activation of STAT6 results in phosphorylation of serine 407 and tyrosine 641. The S407 phosphorylation is unique to virus-induced STAT6 activation and may result in a different binding specificity of STAT6 homodimers.

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Recently, the role of cyclic GMP-AMP (cGAMP) in mediating cellular immunity has been increasingly appreciated. In response to binding of cytosolic DNA, the cGAMP synthase (cGAS) produces the second messenger cGAMP [80, 81]. This compound can then bind to and

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activate STING, leading to IRF3 activation and downstream ISG induction [82]. One of the intriguing features of this pathway is that the second messenger can be transmitted to

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neighbouring cells via gap junctions [83]. This intercellular communication may contribute to

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the antiviral state that has been observed in the absence of secreted cytokines. Recently, cGAS has also been implicated in the control of RNA viruses and thus may have a broader role than

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was originally reported in sensing viral infection of cells [84].

6. Conclusion

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All living organisms have evolved mechanisms to protect themselves from pathogen attack. Mammals and other higher ordered species have evolved to recognize conserved patterns of

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invading pathogens and rapidly produce IFN. The power of rapid IFN production is the

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subsequent induction of hundreds of ISGs, which collectively function to block all aspects of the life cycle of a pathogen. Accordingly, pathogens have co-evolved strategies to inhibit their recognition and subsequent induction of IFN. By blocking key components of the recognition and response pathways, pathogens such as viruses can gain the upper hand and ensure their survival. It is therefore not surprising that cells must have an additional mechanism to sense the danger of an invading pathogen. Indeed, it is becoming increasingly apparent that many generic features of a viral infection, such as membrane perturbation, modulation of the cytoskeleton, and induction of ER stress, are viewed by the cell as a “danger signal”, and lead to the induction, or

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priming, of intrinsic and innate responses. While these responses may involve components of the PAMP-induced IFN response, in many cases, they are modified responses that involve different players. Given that these “danger” signals occur in response to disruption of

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homeostatic cellular processes, one would imagine that it would be more difficult for a pathogen to devise specific counter strategies.

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As such, the IFN-independent innate response may serve as a redundant mechanism in the

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event that a pathogen has successfully blocked IFN production. However, these IFNindependent responses also allow the cell to mount a direct antiviral attack in a cell-restricted

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manner. This restricted response allows the cell to respond to low-level pathogen stimulation without launching a full IFN response, with the associated sequelae elicited by activation of

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hundreds of genes and infiltration of various immune cells. Indeed, the predominantly intracellular nature of this response may be its most salient feature. As a relatively restricted

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phenomenon, an antiviral response in the absence of secreted IFN allows the cell to respond to

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danger signals without committing to the full IFN response. This may allow cells to respond to an unidentified danger signal, through recognition of disrupted homeostasis, by inducing

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expression of PRRs, or activating key transcription factors, thereby priming the cell to further stimulation by PAMPS or DAMPS. In the reports of IFN-independent ISG induction, there is evidence of the up-regulation of the RNA sensors OAS-1 [16], RIG-I [9] and MDA5 [85], as well as the DNA sensor IFI16 [16, 25, 32] and the multi-functional protein tetherin [86]. Having flexibility in inducing intrinsic and innate defense pathways allows a cell to respond appropriately according to the level of danger, as well as sensing that “something isn’t right” and being on alert for potential invasion. Moreover, it arms the cell with additional strategies to counteract the increasingly complex immune evasion strategies encoded by pathogens.

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Conflict of Interests

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The authors declare there is no conflict of interests in this review.

Acknowledgements

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The work in the Mossman laboratory is supported by operating grants from the Canadian

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Institutes for Health Research.

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Fig. 1. Cellular stress pathways activated during viral infection lead to danger sensing and activation of antiviral genes. Activation of innate immune transcription factors (TF) occurs in

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response to induction of cellular stress pathways, which are typically induced during virus

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represent a more generic response to disruption of homeostasis.

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infection. These “danger” signals are often independent of pathogen-associated signals and

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Fig. 2. Localized, intracellular responses protect cells from low-level virus exposure and prime cells for further pathogen attack. IFN-independent cellular responses serve to protect cells in a

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localized fashion, predominantly in response to low-level virus exposure, through the direct induction of ISGs or production and intercellular spread of cGAMP. In addition to rapidly

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protecting primary infected cells from low-level exposure, this response likely serves to prime cells to rapidly and more efficiently respond to subsequent secondary exposure. Under

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surrounding cells and recruitment and activation of immune cells.

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 Cells sense virus infections through pathogen-associated motifs  Prototypic antiviral responses lead to IFN production  Stress pathway induction during infection is a danger signal  Danger responses and IFN-independent responses are localized  Low level responses can prime cells to larger viral challenges

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Highlights

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Susan Collins, M.Sc., is a research technician at McMaster University, in the laboratory of Dr. Karen Mossman. She graduated with an Honours Bachelor of Science in Biochemistry from the University of Guelph in 1998. She subsequently completed a Masters of Science degree at the University of British Columbia in 2001. She currently studies aspects of the host innate immune response following the entry of viral particles. She has contributed to 9 primary research articles within the Mossman laboratory, in the Journal of Virology, Journal of Immunology, Molecular Immunology, PLoS ONE and PLoS Pathogens.

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Karen Mossman, Ph.D., is a Professor in the Department of Pathology and Molecular Medicine and Chair of the Department of Biochemistry and Biomedical Sciences at McMaster University, Canada. She received her Ph.D. in Biochemistry from the University of Alberta, Canada, in 1997. Dr. Mossman’s research focuses on understanding the interactions between viruses and their hosts, both in normal healthy cells and in cancer cells, with the goal of developing novel therapy approaches for emerging viral infections and cancer. Dr. Mossman is the recipient of the Milstein Young Investigator Award and Christina Fleischmann Award from the International Society for Interferon and Cytokine Research. She currently serves as Treasurer of the International Cytokine and Interferon Society and was a co-organizer of the inaugural meeting of the ICIS in San Francisco in 2013. Dr. Mossman has published over 70 peerreviewed scientific publications and has contributed as author and editor to numerous books, including virology textbooks. She serves on the Editorial Board of Journal of Virology, Journal of Interferon and Cytokine Research and PeerJ, and functions as an Associate Editor for Cytokine and Section Editor for PLoS Pathogens.

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