**2. Innate immune response against viral infection**

The mammalian immune system is composed of the innate and the adaptive arms which work in combination to battle against a large variety of pathogens such as bacteria, fungi, parasites

© 2013 Pulido et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of all the acronyms and abbreviations used in the text and figures is shown in Table 1.

**Strategies and Vaccine Improvement** 

2CISA-INIA, Valdeolmos, Madrid

Spain

Spain

msaiz@cbm.uam.es

**1. Introduction** 

Miguel R. Rodríguez Pulido1, Francisco Sobrino1, Belén Borrego2, Margarita Sáiz1

1Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, Madrid

**Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral** 

This chapter will focus on the role of innate immunity induction on antiviral responses with an emphasis on nucleic acids as type-I interferon (IFN) inducers and their use as antiviral compounds and vaccine adjuvants. A general and up-to-date view of the different mechanisms operating in the host cell for sensing viral genomes will be given, as well as viral strategies counteracting this response through immune evasion or specifically targeted antagonism. Our own recent data describing the ability to induce IFN and mediate protection against viral infection in vivo of synthetic RNA transcripts enclosing structural domains present in the 5´ and 3´-terminal regions of the foot-and-mouth disease virus (FMDV) genome will be summarized and discussed in this context. New vaccine formulations including innate immunity inducers are being developed for improvement of current vaccines. The

discussed. A schematic summary of the interrelated topics addressed in this chapter is shown in Figure 1. Additionally, a glossary

extracellular leucine-rich repeats and intracellular Toll/Interleukin-1 receptor (TIR) domain (Figure 2). Nucleic acid sensing-TLRs localize in intracellular vesicles, including endoplasmic reticulum (ER), endosomes, lysosomes and endolysosomes of dendritic cells and other innate immune cells. Intracellular localization enables TLRs to recognize nucleic acids delivered to intracellular compartments after the uptake of viruses or infected cells. Unc93b1 is a membrane protein which anchors TLRs to the ER and regulates their trafficking to the endosomal compartments. TLR signaling pathway depends on the recruitment of a TIR-domain contain‐ ing adaptor, MyD88, for all TLRs (with the exception of TLR3) and culminates with NF-κB and MAP kinase activation and induction of inflammatory cytokines (Figure 2). TLR3 uses TRIF to activate NF-κB and IRF-3 through an alternative pathway and the induction of type-I IFN and inflammatory cytokines. Activation of the TLR signaling leads to maturation of DCs,

Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral Strategies and Vaccine

Improvement

195

http://dx.doi.org/10.5772/56099

The RLRs are a family of ubiquitous cytosolic helicases consisting of the three members: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physi‐ ology-2 (LGP2) (Figure 2). RIG-I and MDA5 have tandem caspase activation and recruitment domains (CARD) followed by a DExD/H box RNA helicase domain and a repressor domain. LGP2 lacks the N-terminal CARD domains and may function to regulate RIG-I and MDA5 as a repressor [11]. It has been reported that RIG-I recognizes ssRNA bearing a 5´-ppp and short dsRNA, while MDA5 senses long dsRNA [12-14]. When the inactive forms of RIG-I or MDA5 bind viral RNA, the helicases undergo a conformational change, multimerization and then, interaction with the adaptor molecule MAVS (also called IPS1, VISA or CARDIF), localised to the outer mitochondrial membrane via CARD-CARD interaction. Then, MAVS induces activation of IRF3/7 resulting in the transcription of type-I IFNs and also activates NF-κB (Figure 2). It has been recently shown that MAVS resides on peroxisomes also and can induce antiviral signaling from this organelle acting with mitochondrial MAVS sequentially to create an antiviral cellular state [15]. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors for short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed

In addition to PRRs, which inhibit viral infections indirectly by activating signaling cascades that result in the transcription of IFN and other antiviral molecules, there are intrinsic antiviral factors which act blocking viral replication immediately and directly, often before the onset of IFN response, like PKR, MxA, TRIM5α or the IFIT and IFITM families [16]. Intrinsic innate factors preexist in certain cell types though they can be further induced by IFNs to amplify

Recent work supports the non-redundant functional requirement for TLRs and RLRs [17]. On the contrary, the cooperation and crosstalk between different PRRs mediates activation of an effective immune response and host defense against viral infections [8, 18]. Unique links between NLRs and RLRs signaling responses have also been identified [10, 19]. Polymicrobial infection involve complex host interactions that are likely to engage a variety of response

contributing to the induction of adaptive immunity.

kinetics, amplifying and stabilizing the antiviral response.

their antiviral activity.

pathways including different PRRs.

**Figure 1.** Schematic summary of the topics reviewed and discussed in this chapter

and viruses. Both systems have the molecular task to distinguish "self" from "non-self" components in the organisms in a sensitive and faithful manner. The innate immune system is hence the first line of defense against infection by pathogens. The existence of patternrecognition receptors (PRRs) expressed in cells of the innate immune system that are capable to specifically sense pathogen-associated molecular patterns (PAMPs) was first proposed by Charles Janeway in 1989 [1]. PAMPs are chemical or structural features present in pathogens but not in host cells acting then as alert signals to the innate immune system of the host. Lipids, polysaccharides, nucleic acids and CpG DNA are among the basic PAMPs recognized by PRRs. Their recognition triggers cellular responses aimed to counteract the pathogen and initiate and promote other responses such as inflammation and adaptive immune responses. Figure 1. Schematic summary of the topics reviewed and discussed in this chapter **2. Innate immune response against viral infection**  The mammalian immune system is composed of the innate and the adaptive arms which work in combination to battle against a large variety of pathogens such as bacteria, fungi, parasites and viruses. Both systems have the molecular task to distinguish "self" from "non-self" components in the organisms in a sensitive and faithful manner. The innate immune system is hence the first line of defense against infection by pathogens. The existence of pattern-recognition receptors (PRRs) expressed in cells of the innate immune system that are capable to specifically sense pathogen-associated molecular patterns (PAMPs) was first proposed by Charles Janeway in 1989 [1]. PAMPs are chemical or structural features present in pathogens but not in host cells acting then as alert signals to the innate immune system of the host. Lipids, polysaccharides, nucleic acids and CpG DNA are among the basic

**Figure 1.** Schematic summary of the topics reviewed and discussed in this chapter

PAMPs recognized by PRRs. Their recognition triggers cellular responses aimed to counteract the pathogen and initiate and

In 1996, the gene/protein Toll, initially described as a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo, was found to play also a role in immunity against fungal infections [2]. One year later, their mammalian orthologs, the Toll-like receptors (TLRs), were reported to mediate recognition of pathogens by the innate immune system [3]. In 2004, a new and TLR-independent pathway was described for recognition of viral nucleic acids in the cytoplasm of the infected cells, the retinoic acid-inducible gene-1 (RIG-I) [4]. Four different families of PRRs have been found to date, including TLRs [5], RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) [6] and nucleotide oligomerization domain (NOD)-like receptors, (NLRs) [7], as well as unidentified proteins that mediate sensing of cytosolic DNA or retroviral infection [8]. Among them, TLRs, RLRs and NLRs are involved in the recognition of viral nucleic acids [9]. NOD2, a member of the NLR family, typically involved in antimi‐ crobial immune defenses, and highly expressed in antigen presenting cells (APCs) such as monocytes, macrophages and dendritic cells (DCs), has been shown to bind viral singlestranded (ss) RNA [10]. promote other responses such as inflammation and adaptive immune responses.

TLRs are a family of type-I transmembrane proteins that traffic between the plasma membrane and endosomal vesicles, expressed on various immune cells, including dendritic cells, which recognize a wide range of PAMPs including double-stranded (ds) RNA (TLR3), ssRNA (TLR7 and TLR8) and DNA (TLR9). All TLRs signal as dimmers and share a common architecture of extracellular leucine-rich repeats and intracellular Toll/Interleukin-1 receptor (TIR) domain (Figure 2). Nucleic acid sensing-TLRs localize in intracellular vesicles, including endoplasmic reticulum (ER), endosomes, lysosomes and endolysosomes of dendritic cells and other innate immune cells. Intracellular localization enables TLRs to recognize nucleic acids delivered to intracellular compartments after the uptake of viruses or infected cells. Unc93b1 is a membrane protein which anchors TLRs to the ER and regulates their trafficking to the endosomal compartments. TLR signaling pathway depends on the recruitment of a TIR-domain contain‐ ing adaptor, MyD88, for all TLRs (with the exception of TLR3) and culminates with NF-κB and MAP kinase activation and induction of inflammatory cytokines (Figure 2). TLR3 uses TRIF to activate NF-κB and IRF-3 through an alternative pathway and the induction of type-I IFN and inflammatory cytokines. Activation of the TLR signaling leads to maturation of DCs, contributing to the induction of adaptive immunity.

The RLRs are a family of ubiquitous cytosolic helicases consisting of the three members: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physi‐ ology-2 (LGP2) (Figure 2). RIG-I and MDA5 have tandem caspase activation and recruitment domains (CARD) followed by a DExD/H box RNA helicase domain and a repressor domain. LGP2 lacks the N-terminal CARD domains and may function to regulate RIG-I and MDA5 as a repressor [11]. It has been reported that RIG-I recognizes ssRNA bearing a 5´-ppp and short dsRNA, while MDA5 senses long dsRNA [12-14]. When the inactive forms of RIG-I or MDA5 bind viral RNA, the helicases undergo a conformational change, multimerization and then, interaction with the adaptor molecule MAVS (also called IPS1, VISA or CARDIF), localised to the outer mitochondrial membrane via CARD-CARD interaction. Then, MAVS induces activation of IRF3/7 resulting in the transcription of type-I IFNs and also activates NF-κB (Figure 2). It has been recently shown that MAVS resides on peroxisomes also and can induce antiviral signaling from this organelle acting with mitochondrial MAVS sequentially to create an antiviral cellular state [15]. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors for short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, amplifying and stabilizing the antiviral response.

and viruses. Both systems have the molecular task to distinguish "self" from "non-self" components in the organisms in a sensitive and faithful manner. The innate immune system is hence the first line of defense against infection by pathogens. The existence of patternrecognition receptors (PRRs) expressed in cells of the innate immune system that are capable to specifically sense pathogen-associated molecular patterns (PAMPs) was first proposed by Charles Janeway in 1989 [1]. PAMPs are chemical or structural features present in pathogens but not in host cells acting then as alert signals to the innate immune system of the host. Lipids, polysaccharides, nucleic acids and CpG DNA are among the basic PAMPs recognized by PRRs. Their recognition triggers cellular responses aimed to counteract the pathogen and initiate and

**Figure 1.** Schematic summary of the topics reviewed and discussed in this chapter

*Viral immune evasion strategies*

The mammalian immune system is composed of the innate and the adaptive arms which work in combination to battle against a large variety of pathogens such as bacteria, fungi, parasites and viruses. Both systems have the molecular task to distinguish "self" from "non-self" components in the organisms in a sensitive and faithful manner. The innate immune system is hence the first line of defense against infection by pathogens. The existence of pattern-recognition receptors (PRRs) expressed in cells of the innate immune system that are capable to specifically sense pathogen-associated molecular patterns (PAMPs) was first proposed by Charles Janeway in 1989 [1]. PAMPs are chemical or structural features present in pathogens but not in host cells acting then as alert signals to the innate immune system of the host. Lipids, polysaccharides, nucleic acids and CpG DNA are among the basic PAMPs recognized by PRRs. Their recognition triggers cellular responses aimed to counteract the pathogen and initiate and

**Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral** 

This chapter will focus on the role of innate immunity induction on antiviral responses with an emphasis on nucleic acids as type-I interferon (IFN) inducers and their use as antiviral compounds and vaccine adjuvants. A general and up-to-date view of the different mechanisms operating in the host cell for sensing viral genomes will be given, as well as viral strategies counteracting this response through immune evasion or specifically targeted antagonism. Our own recent data describing the ability to induce IFN and mediate protection against viral infection in vivo of synthetic RNA transcripts enclosing structural domains present in the 5´ and 3´-terminal regions of the foot-and-mouth disease virus (FMDV) genome will be summarized and discussed in this context. New vaccine formulations including innate immunity inducers are being developed for improvement of current vaccines. The potential of exogenous nucleic acids as modulators of immune response outcomes and vaccine adjuvants will be reviewed and discussed. A schematic summary of the interrelated topics addressed in this chapter is shown in Figure 1. Additionally, a glossary

*Synthetic molecules*

*RLRs/TLRs ligands*

*Application to new therapeutic, vaccine and antiviral developments*

*Review, discussion and future perspectives*

*RNA transcripts mimicking structural motifs in the FMDV genome*

**Strategies and Vaccine Improvement** 

2CISA-INIA, Valdeolmos, Madrid

Spain

Spain

msaiz@cbm.uam.es

**1. Introduction** 

*Viral infection*

*Host antiviral response*

Miguel R. Rodríguez Pulido1, Francisco Sobrino1, Belén Borrego2, Margarita Sáiz1

of all the acronyms and abbreviations used in the text and figures is shown in Table 1.

194 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

1Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, Madrid

In 1996, the gene/protein Toll, initially described as a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo, was found to play also a role in immunity against fungal infections [2]. One year later, their mammalian orthologs, the Toll-like receptors (TLRs), were reported to mediate recognition of pathogens by the innate immune system [3]. In 2004, a new and TLR-independent pathway was described for recognition of viral nucleic acids in the cytoplasm of the infected cells, the retinoic acid-inducible gene-1 (RIG-I) [4]. Four different families of PRRs have been found to date, including TLRs [5], RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) [6] and nucleotide oligomerization domain (NOD)-like receptors, (NLRs) [7], as well as unidentified proteins that mediate sensing of cytosolic DNA or retroviral infection [8]. Among them, TLRs, RLRs and NLRs are involved in the recognition of viral nucleic acids [9]. NOD2, a member of the NLR family, typically involved in antimi‐ crobial immune defenses, and highly expressed in antigen presenting cells (APCs) such as monocytes, macrophages and dendritic cells (DCs), has been shown to bind viral single-

TLRs are a family of type-I transmembrane proteins that traffic between the plasma membrane and endosomal vesicles, expressed on various immune cells, including dendritic cells, which recognize a wide range of PAMPs including double-stranded (ds) RNA (TLR3), ssRNA (TLR7 and TLR8) and DNA (TLR9). All TLRs signal as dimmers and share a common architecture of

promote other responses such as inflammation and adaptive immune responses.

promote other responses such as inflammation and adaptive immune responses.

Figure 1. Schematic summary of the topics reviewed and discussed in this chapter

*Innate response*

**Figure 1.** Schematic summary of the topics reviewed and discussed in this chapter

*Host cell PRRs*

*PAMP motifs in the viral genome*

**2. Innate immune response against viral infection** 

stranded (ss) RNA [10].

In addition to PRRs, which inhibit viral infections indirectly by activating signaling cascades that result in the transcription of IFN and other antiviral molecules, there are intrinsic antiviral factors which act blocking viral replication immediately and directly, often before the onset of IFN response, like PKR, MxA, TRIM5α or the IFIT and IFITM families [16]. Intrinsic innate factors preexist in certain cell types though they can be further induced by IFNs to amplify their antiviral activity.

Recent work supports the non-redundant functional requirement for TLRs and RLRs [17]. On the contrary, the cooperation and crosstalk between different PRRs mediates activation of an effective immune response and host defense against viral infections [8, 18]. Unique links between NLRs and RLRs signaling responses have also been identified [10, 19]. Polymicrobial infection involve complex host interactions that are likely to engage a variety of response pathways including different PRRs.

IFNs exert auto- and paracrine actions within a few hours in response to a viral infection. Their protective effect is dual: they induce an antiviral cellular state and promote the clearance of infected cells in synergy with other proapoptotic agents as tumor necrosis factor (TNF). Through the secretion of IFN, triggered by activation and translocation to the nucleus of NFκB, IRF3 and IRF7, the antiviral response can be amplified and spread to surrounding uninfected cells by binding to the IFN-α/β receptor (IFNAR) in the cell surface. Binding of the cytokine triggers a Jak-STAT signaling pathway and subsequently activates hundreds of IFNstimulated genes (ISGs), most of them encoding proteins with antiviral functions such as inhibition of viral gene expression or degradation of the viral genome [20].

Induction of innate immune signaling pathways through PRRs is a crucial step antagonised by many viruses [24, 25]. Over 170 different virus-encoded IFN antagonists from 93 different viruses had been reported by 2010 (reviewed in [26]), and the list keeps constantly growing, indicating that most viruses interfere with multiple stages of the IFN response. Four main mechanisms are used to circumvent host innate responses: general inhibition of gene expres‐ sion, and sequestration, proteoltytic cleavage or proteasome degradation of key factors of the IFN circuit such as RLRs, MAVS, IRFs, Jak/STAT, PKR... Several IFN antagonists are conserved within different RNA virus families, while that seems not to be the case for DNA viruses [26]. This can be explained by the multi-functionality of RNA virus proteins, imposed by restriction in genome size, unlike large dsDNA viruses which might have a higher coding capacity for

Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral Strategies and Vaccine

Improvement

197

http://dx.doi.org/10.5772/56099

The potential of IFN antagonists knockout viruses as promising candidates for live virus vaccines has been suggested based on studies with Influenza A/B viruses, Japanese encepha‐ litis virus, human respiratory syncytial virus and coronaviruses [26-29]. These severely attenuated viruses are rapidly cleared in vivo by a potent IFN response, while inducing longlasting immune memory due to their replication competent nature. Viral miRNAs may also function in evasion of the host antiviral response (reviewed in [30]). The contribution to viral evasion of type-I IFN response of small non-coding subgenomic flavivirus RNAs generated as degradation products by a host exoribonuclease, has been recently shown [31]. Hence, IFN

new viral proteins displaying a wider range of antagonistic activities.

antagonists are good targets for antiviral drugs development.

accurate PAMP discrimination [33].

**3. RNA motifs in the viral genome can trigger innate responses**

tidylate-phosphate-deoxyguanylate (CpG) motifs in viral and bacterial DNA [37].

Accurate discrimination of self from non-self is critical to avoid immune triggering against self that leads to autoimmunity [32]. In that sense, it has been proposed for hepatitis C virus (HCV) that a combinatorial non-self signature in the viral genome for PRR binding may lead to

TLRs involved in recognition of the viral genomes are TLR3, TLR7/8 and TLR9, all of them localized to the endosomal compartment [34]. TLR3 is widely expressed in innate immune cells with the exception of neutrophils and pDCs and responds to dsRNA, a common viral PAMP, and its synthetic analog polyriboinosinic-polyribocytidylic acid (poly I:C) [35]. TLR7 and TLR8 are closely related receptors that recognize nearly any long ssRNA with some differences between them. Short ssRNA containing certain motifs preferentially activate TLR7, and activation with synthetic agonists specific to TLR7 or TLR8 trigger different cytokine profiles [36]. TLR9 is highly expressed in pDCs and responds to the unmethylated deoxycy‐

Different features have been defined for RIG-I recognition as RNA PAMPs, including the presence of a free 5´-triphosphate, absent from eukaryotic cytoplasm due to RNA metabolism in the nucleus, length (longer than 19 nt), secondary structure characteristics (a base-pairing region of 10-20 nt near the 5´-ppp) [38] and nucleotide sequence motifs (such as a 3´-poly U/UC tract in the HCV genome) [33]. Panhandle structures adopted by Sendai virus DI-

In addition to its antiviral properties, IFNs exhibit potent immunomodulatory properties that contribute to their antiviral effect such as stimulation of the effector function of natural killer (NK) cells, cytotoxic T lymphocytes and macrophages, upregulation of MHC class I and II molecules, induction of immunoglobulin production by B cells and stimulation of proliferation of memory T cells [21]. This enables several ways to control viral replication by modulating of the innate and adaptive immune responses [22]. Type-I IFNs act through activation and maturation of dendritic cells leading to MHC upregulation. They can also regulate certain chemokines, chemokine receptors and costimulatory molecules, which, in turn, stimulate CD4 and CD8-positive T cell responses and promote Th1 differentiation, modulating T lymphocyte responses [23].

**Figure 2.** Schematic representation of viral RNA detection by TLRs and RLRs and signaling patways

Figure 2. Schematic representation of viral RNA detection by TLRs and RLRs and signalling pathways

**3. RNA motifs in the viral genome can trigger innate responses** 

binding may lead to accurate PAMP discrimination [33].

deoxyguanylate (CpG) motifs in viral and bacterial DNA [37].

antiviral drugs development.

have a higher coding capacity for new viral proteins displaying a wider range of antagonistic activities.

Induction of innate immune signaling pathways through PRRs is a crucial step antagonised by many viruses [24, 25]. Over 170 different virus-encoded IFN antagonists from 93 different viruses had been reported by 2010 (reviewed in [26]), and the list keeps constantly growing, indicating that most viruses interfere with multiple stages of the IFN response. Four main mechanisms are used to circumvent host innate responses: general inhibition of gene expression, and sequestration, proteoltytic cleavage or proteasome degradation of key factors of the IFN circuit such as RLRs, MAVS, IRFs, Jak/STAT, PKR... Several IFN antagonists are conserved within different RNA virus families, while that seems not to be the case for DNA viruses [26]. This can be explained by the multi-functionality of RNA virus proteins, imposed by restriction in genome size, unlike large dsDNA viruses which might

The potential of IFN antagonists knockout viruses as promising candidates for live virus vaccines has been suggested based on studies with Influenza A/B viruses, Japanese encephalitis virus, human respiratory syncytial virus and coronaviruses [26-29]. These severely attenuated viruses are rapidly cleared in vivo by a potent IFN response, while inducing long-lasting immune memory due to their replication competent nature. Viral miRNAs may also function in evasion of the host antiviral response (reviewed in [30]). The contribution to viral evasion of type-I IFN response of small non-coding subgenomic flavivirus RNAs generated as degradation products by a host exoribonuclease, has been recently shown [31]. Hence, IFN antagonists are good targets for

Accurate discrimination of self from non-self is critical to avoid immune triggering against self that leads to autoimmunity [32]. In that sense, it has been proposed for hepatitis C virus (HCV) that a combinatorial non-self signature in the viral genome for PRR

TLRs involved in recognition of the viral genomes are TLR3, TLR7/8 and TLR9, all of them localized to the endosomal compartment [34]. TLR3 is widely expressed in innate immune cells with the exception of neutrophils and pDCs and responds to dsRNA, a common viral PAMP, and its synthetic analog polyriboinosinic-polyribocytidylic acid (poly I:C) [35]. TLR7 and TLR8 are closely related receptors that recognize nearly any long ssRNA with some differences between them. Short ssRNA containing certain motifs preferentially activate TLR7, and activation with synthetic agonists specific to TLR7 or TLR8 trigger different cytokine profiles [36]. TLR9 is highly expressed in pDCs and responds to the unmethylated deoxycytidylate-phosphate-

Different features have been defined for RIG-I recognition as RNA PAMPs, including the presence of a free 5´-triphosphate, absent from eukaryotic cytoplasm due to RNA metabolism in the nucleus, length (longer than 19 nt), secondary structure characteristics (a Induction of innate immune signaling pathways through PRRs is a crucial step antagonised by many viruses [24, 25]. Over 170 different virus-encoded IFN antagonists from 93 different viruses had been reported by 2010 (reviewed in [26]), and the list keeps constantly growing, indicating that most viruses interfere with multiple stages of the IFN response. Four main mechanisms are used to circumvent host innate responses: general inhibition of gene expres‐ sion, and sequestration, proteoltytic cleavage or proteasome degradation of key factors of the IFN circuit such as RLRs, MAVS, IRFs, Jak/STAT, PKR... Several IFN antagonists are conserved within different RNA virus families, while that seems not to be the case for DNA viruses [26]. This can be explained by the multi-functionality of RNA virus proteins, imposed by restriction in genome size, unlike large dsDNA viruses which might have a higher coding capacity for new viral proteins displaying a wider range of antagonistic activities.

IFNs exert auto- and paracrine actions within a few hours in response to a viral infection. Their protective effect is dual: they induce an antiviral cellular state and promote the clearance of infected cells in synergy with other proapoptotic agents as tumor necrosis factor (TNF). Through the secretion of IFN, triggered by activation and translocation to the nucleus of NFκB, IRF3 and IRF7, the antiviral response can be amplified and spread to surrounding uninfected cells by binding to the IFN-α/β receptor (IFNAR) in the cell surface. Binding of the cytokine triggers a Jak-STAT signaling pathway and subsequently activates hundreds of IFNstimulated genes (ISGs), most of them encoding proteins with antiviral functions such as

In addition to its antiviral properties, IFNs exhibit potent immunomodulatory properties that contribute to their antiviral effect such as stimulation of the effector function of natural killer (NK) cells, cytotoxic T lymphocytes and macrophages, upregulation of MHC class I and II molecules, induction of immunoglobulin production by B cells and stimulation of proliferation of memory T cells [21]. This enables several ways to control viral replication by modulating of the innate and adaptive immune responses [22]. Type-I IFNs act through activation and maturation of dendritic cells leading to MHC upregulation. They can also regulate certain chemokines, chemokine receptors and costimulatory molecules, which, in turn, stimulate CD4 and CD8-positive T cell responses and promote Th1 differentiation, modulating T lymphocyte

**TLR7/8**

**Proinflammatory cytokines transcription**

**Figure 2.** Schematic representation of viral RNA detection by TLRs and RLRs and signaling patways

**MyD88 MyD88 TRIF**

*Nucleus*

**TLR9 DNA**

inhibition of viral gene expression or degradation of the viral genome [20].

196 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Figure 2. Schematic representation of viral RNA detection by TLRs and RLRs and signalling pathways

**Figure 2.** Schematic representation of viral RNA detection by TLRs and RLRs and signalling pathways

**3. RNA motifs in the viral genome can trigger innate responses** 

binding may lead to accurate PAMP discrimination [33].

deoxyguanylate (CpG) motifs in viral and bacterial DNA [37].

have a higher coding capacity for new viral proteins displaying a wider range of antagonistic activities.

Induction of innate immune signaling pathways through PRRs is a crucial step antagonised by many viruses [24, 25]. Over 170 different virus-encoded IFN antagonists from 93 different viruses had been reported by 2010 (reviewed in [26]), and the list keeps constantly growing, indicating that most viruses interfere with multiple stages of the IFN response. Four main mechanisms are used to circumvent host innate responses: general inhibition of gene expression, and sequestration, proteoltytic cleavage or proteasome degradation of key factors of the IFN circuit such as RLRs, MAVS, IRFs, Jak/STAT, PKR... Several IFN antagonists are conserved within different RNA virus families, while that seems not to be the case for DNA viruses [26]. This can be explained by the multi-functionality of RNA virus proteins, imposed by restriction in genome size, unlike large dsDNA viruses which might

*Cytoplasm*

**Tyk2 Jak1**

*JAK/STAT pathway*

**IFN‐/β**

**IRF‐9**

**P P STAT‐<sup>2</sup> STAT‐1**

**ISG transcription**

**Virus infection IFNAR 1/2**

*Endosome*

**TLR3 dsRNA ssRNA**

The potential of IFN antagonists knockout viruses as promising candidates for live virus vaccines has been suggested based on studies with Influenza A/B viruses, Japanese encephalitis virus, human respiratory syncytial virus and coronaviruses [26-29]. These severely attenuated viruses are rapidly cleared in vivo by a potent IFN response, while inducing long-lasting immune memory due to their replication competent nature. Viral miRNAs may also function in evasion of the host antiviral response (reviewed in [30]). The contribution to viral evasion of type-I IFN response of small non-coding subgenomic flavivirus RNAs generated as degradation products by a host exoribonuclease, has been recently shown [31]. Hence, IFN antagonists are good targets for

Accurate discrimination of self from non-self is critical to avoid immune triggering against self that leads to autoimmunity [32]. In that sense, it has been proposed for hepatitis C virus (HCV) that a combinatorial non-self signature in the viral genome for PRR

TLRs involved in recognition of the viral genomes are TLR3, TLR7/8 and TLR9, all of them localized to the endosomal compartment [34]. TLR3 is widely expressed in innate immune cells with the exception of neutrophils and pDCs and responds to dsRNA, a common viral PAMP, and its synthetic analog polyriboinosinic-polyribocytidylic acid (poly I:C) [35]. TLR7 and TLR8 are closely related receptors that recognize nearly any long ssRNA with some differences between them. Short ssRNA containing certain motifs preferentially activate TLR7, and activation with synthetic agonists specific to TLR7 or TLR8 trigger different cytokine profiles [36]. TLR9 is highly expressed in pDCs and responds to the unmethylated deoxycytidylate-phosphate-

Different features have been defined for RIG-I recognition as RNA PAMPs, including the presence of a free 5´-triphosphate, absent from eukaryotic cytoplasm due to RNA metabolism in the nucleus, length (longer than 19 nt), secondary structure characteristics (a

responses [23].

*In vitro* **transcripts**

**5'‐PPP**

**LGP‐2**

**RIG‐1 MAD‐5**

**5'‐PPP**

*Viral RNA*

**MAVS**

*Mitochondrion*

**NF‐kB**

**IRF‐3**

**IRF‐7 P P**

**Type‐I IFNs transcription**

**long poly(I:C) short poly(I:C)**

*Replication intermediates*

antiviral drugs development.

The potential of IFN antagonists knockout viruses as promising candidates for live virus vaccines has been suggested based on studies with Influenza A/B viruses, Japanese encepha‐ litis virus, human respiratory syncytial virus and coronaviruses [26-29]. These severely attenuated viruses are rapidly cleared in vivo by a potent IFN response, while inducing longlasting immune memory due to their replication competent nature. Viral miRNAs may also function in evasion of the host antiviral response (reviewed in [30]). The contribution to viral evasion of type-I IFN response of small non-coding subgenomic flavivirus RNAs generated as degradation products by a host exoribonuclease, has been recently shown [31]. Hence, IFN antagonists are good targets for antiviral drugs development.
