**4. Viral alternatives to cap structures**

predicted to be at the MTase active site. The 2'O position of the GpppN is methylated prior to the guanine N7 position, which is the opposite order when compared to most canonical cap-1 methylation events [33, 53]. The overall GDP RNA capping sequence can be summarized as NTPase→PRNTase→2'OMTase→N7MTase (Fig. 7). It is very likely that an ancestral (+)ssRNA virus polymerase has evolved a PRNTase activity independently from its eukaryotic host. Both

Some viruses, unable to synthesize their own cap structures, have evolved a clever way to acquire this important entity: steeling it from their host. This method of cap acquisition, termed RNA cap snatching, is used by representatives of the *Orthomyxoviridae* (e.g. Influenza virus, Thogoto virus), the *Arenaviridae* (e.g. Lassa virus, Machupo virus) and the *Bunyaviridae* (Hantaan virus, La Crosse virus, Tomato Spotted Wilt virus) families [5, 58]. These (-)ssRNA viruses acquire their cap structure from their hosts capped mRNA. They bind the cap structure, cleave the RNA a few nucleotides downstream and finally use this short capped RNA to prime their RDRP [59]. The *Arenaviridae* and *Bunyaviridae* express a large monomeric polymerase where the *Orthomyxoviridae* expresses an heterotrimeric polymerase (e.g. PB1, PB2 and PA protein of influenza virus) harbouring all the activities required for cap snatching. The PB2 protein of the Influenza virus, the most studied cap snatching virus, specifically binds the host mRNA cap structure. The specificity of the binding is crucial and is mediated by the aromatic stacking of the methylated gunanine coupled to a base-specific interaction with a conserved acidic residue [60]. While the mode of cap binding is similar between PB2 and other capbinding proteins (e.g. eIF4E, nuclear cap binding complex, Vaccinia VP39) its overall fold is completely different [60]. Once the host mRNA is bound by the cap-binding PB2, the viral PA subunit cleaves the mRNA a few nucleotides downstream from the cap structure. The length of the primer RNA generated is virus-dependent, and typically ranges from 10-13 nucleotides for Influenza virus, but can be as short as 1-2 nucleotides as is seen in the Thogoto virus [59, 61, 62]. The PA endonuclease domain shares a high homology with the type II restriction enzyme, including the active site conserved (P)Dxn(D/E)xK signature motif [63]. The PA active site coordinates two Mn2+ cations and is believed to catalyze endonucleolytic cleavage through a common two-metal dependent mechanism [61, 64]. The short capped oligomers are next used by the PB1 RDRP as primer to initiate the transcription of the viral mRNAs [58]. PB1 also specifically binds the viral RNA (vRNA) 3' and 5'-end through a ribonucleoprotein 1-like motif ((R/K)G(F/Y)(G/A)(F/Y)Vx(F/Y)) [65]. The vRNA serves as a template for the 3' elongation of the cellular 10-13 nucleotide-capped primer. The overall cap snatching process results in the transcription of a chimeric full-length vRNA with a 5'-extension of 10-13 cellular nucleotides and a cap-2 structure (Fig. 8). Cap snatching enables viruses to acquire their hosts cap structure, which not only promotes viral replication but also impairs cellular mRNA translation, as translation of decapped cellular mRNA is impeded and the mRNA is targeted for degradation. Another consequence of cap snatching is the dependency on a pool of host mRNA molecules in order to support viral replication. (-)ssRNA viruses that utilize cap snatching have evolved ways to maintain the precious pool of eukaryotic mRNA. First, the cap binding and endonu‐ clease activity of the trimeric polymerase are only activated upon vRNA binding, limiting the

N7 and 2'OMTase, however, have likely been acquired from a eukaryotic host.

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

**3.9. The RNA cap snatching**

Most viruses harbour a cap structure at the 5'-end of their RNA. Mutations preventing the proper capping of their RNA result in infection or replication deficient viruses. This is a strong proof of the crucial importance of the cap structure for viral RNA stability and translation. Yet not all viruses harbour capped RNA, which raises the question about the mechanism they evolved to overcome this cap dependency? To answer this query it's important to ask whether it is the cap structure itself or its function that is essential. In fact, the cap structure is important for a number of different cellular processes related to mRNA metabolism. For instance, the cap structure protects the RNA from 5'→3' exonucleases, preventing their degradation. The RNA cap structure also represents a definite molecular structure that is specifically recognized by the eukaryotic initiation factor 4E (eIF4E), which, together with the scaffold protein eIF4G, the RNA helicase eIF4A and the ribosome binding protein eIF3, promote RNA translation initiation. While most viruses use a cap structure to fulfill these important roles, some viruses have evolved cap-independent strategies to ensure the stability and translation of their RNA.

#### **4.1. Viral proteins as substitutes for the cap structure**

Viruses of the *Picornaviridae* (e.g. Poliovirus, Hepatitis A virus), *Potyviridae* and *Caliciviridae* (e.g. Norwalk virus, Feline calicivirus) families bear a special type of RNA 5'-end modification. The RNA 5'end of these (+)ssRNA viruses is covalently linked to a viral protein [68]. This viral genome-linked protein (VPg) is not added to the viral genome upon replication, like a regular cap structure, but is instead directly used by the RDRP as a primer to initiate RNA polymeri‐ sation. VPg is a representative of the class II nucleic acid-protein complex and does not catalyze its own covalent complex formation (like GTase or PRNTase could do) [69]. The VPg-RNA formation is instead catalyzed by a second protein, the viral RDRP, which synthesizes the primer in a template-dependent matter, resulting in a virus specific initiating primer, VPgpUpU for *Picornaviridae* and VPg-pGpU for *Calicivirus*[70]. VPg is covalently linked to the first RNA nucleotide via a phosphodiester bond between the RNA α-phosphate and the tyrosine hydroxyl group situated in the conserved motif (E/D)EYDE(Y/W/F)[71]. The VPg protein protects the vRNA 5'-end from the cellular 5'→3' exounucleases, thus limiting the vRNA degradation. Furthermore, the VPg is used to initiate the RNA polymerisation instead of being added once the RNA is synthesised. This prevents the formation of 5'-triphosphate vRNA and limits the cellular anti-viral response, which will be described later [68]. In addition to their protective role against RNA degradation, some VPg can fulfill a second important role of the cap structure, promoting the vRNA translation initiation. This is the case of the *Caliciviridae* and *Potyviridae* 15 kDA VPg that is essential for vRNA translation initiation. This VPg directly interacts with eIF4E (the cap-binding protein) and the eIF3 complex (the 40S binding complex), which promotes the assembly of the translation initiation complex to the 5'-end of the vRNA (Fig. 9) [68, 72-75]. This allows VPg-vRNA to bypass the requirement for a direct eIF4E-cap interaction in order to initiate translation. This property is not conserved among all VPg, the *Picornaviridae* VPg is much smaller (2.5 kDA) and is not involved in the vRNA translation initiation [68]. These viruses instead rely on a highly structured RNA sequence called an internal ribosome entry site (IRES) to ensure their translation (this will also be described in more detail later on). All the (+)ssRNA viruses encoding a VPg benefit from its protective effect on the viral genome, but the *Caliciviridae* and *Potyviridae* VPg have evolved an additional function, promoting vRNA translation initiation. This VPs is a striking example of a cap substitute as it fulfills two critical functions of the cap structure, namely ensuring vRNA stability and promoting translation initiation.

**Figure 9. Mechanisms of viral translation initiation**. The cap structure of RNA is specifically recognized by eIF4E, which recruits the cap-dependent translation initiation complex to the 5'-end of capped RNA. Alternatively, the viral VPg (covalently linked at the 5'-end of vRNA) can directly recruit eIF4E. The conserved viral RNA structure, located within the 5'-UTR, can directly promote translation initiation. These internal ribosome entry sites (IRES) are catego‐ rized into four different groups, each able to directly recruit a subset of initiation factors and the 40S ribosomal subu‐ nit in order to initiate translation. Of notice, some mechanisms of initiation require RNA scanning (left panel) by the initiation complex to reach the ORF while others position the ribosome directly adjacent to the transcription initiation site (right panel). Abbreviation : Human Immunodeficiency virus (HIV), West Nile virus (WNV), Encephalomyocarditis

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The ribonucleic acid (RNA) is a macromolecule which, according to the central dogma of molecular biology, is a transient messenger carrying the genetic information required to pilot the protein synthesis. In addition to this canonical role, RNA, given its high chemical com‐ plexity, can fulfill additional roles including genome support, ordered three-dimensional structure and even catalytic activity [76]. Many viruses have exploited this capacity of RNA to form complex structure in order to promote viral replication. Some viruses, lacking enzymatic activity to synthesize or acquire a cap structure at the 5'-end of their vRNA, have instead selected a high-order structural RNA element upstream of their coding region. This peculiar RNA sequence can fold precisely and repeatedly into a definite three-dimensional structure. This ordered structure has numerous functions including binding to other macro‐ molecule partners. Those viruses use this *cis*-acting structure to bind directly or indirectly to ribosomal components in order to assemble the translation initiation complex at the beginning of their open reading frame (ORF). This promotes the cap-independent translation of viral genes. Such RNA structures bypassing the cap-dependency for translation initiation are called internal ribosome entry site (IRES). Many RNA virus families (e.g. *Dicistroviridae*, *Picornaviri‐ dae* and some *Flaviviridae*) use this structure to promote viral protein production. The diversity

virus (ECMV), Hepatitis C virus (HCV) and Cricket paralysis virus (CPV), Open reading frame (ORF).

**4.2. Highly structured 5' RNA structure as an alternative to the cap structure**

not all viruses harbour capped RNA, which raises the question about the mechanism they evolved to overcome this cap dependency? To answer this query it's important to ask whether it is the cap structure itself or its function that is essential. In fact, the cap structure is important for a number of different cellular processes related to mRNA metabolism. For instance, the cap structure protects the RNA from 5'→3' exonucleases, preventing their degradation. The RNA cap structure also represents a definite molecular structure that is specifically recognized by the eukaryotic initiation factor 4E (eIF4E), which, together with the scaffold protein eIF4G, the RNA helicase eIF4A and the ribosome binding protein eIF3, promote RNA translation initiation. While most viruses use a cap structure to fulfill these important roles, some viruses have evolved cap-independent strategies to ensure the stability and translation of their RNA.

Viruses of the *Picornaviridae* (e.g. Poliovirus, Hepatitis A virus), *Potyviridae* and *Caliciviridae* (e.g. Norwalk virus, Feline calicivirus) families bear a special type of RNA 5'-end modification. The RNA 5'end of these (+)ssRNA viruses is covalently linked to a viral protein [68]. This viral genome-linked protein (VPg) is not added to the viral genome upon replication, like a regular cap structure, but is instead directly used by the RDRP as a primer to initiate RNA polymeri‐ sation. VPg is a representative of the class II nucleic acid-protein complex and does not catalyze its own covalent complex formation (like GTase or PRNTase could do) [69]. The VPg-RNA formation is instead catalyzed by a second protein, the viral RDRP, which synthesizes the primer in a template-dependent matter, resulting in a virus specific initiating primer, VPgpUpU for *Picornaviridae* and VPg-pGpU for *Calicivirus*[70]. VPg is covalently linked to the first RNA nucleotide via a phosphodiester bond between the RNA α-phosphate and the tyrosine hydroxyl group situated in the conserved motif (E/D)EYDE(Y/W/F)[71]. The VPg protein protects the vRNA 5'-end from the cellular 5'→3' exounucleases, thus limiting the vRNA degradation. Furthermore, the VPg is used to initiate the RNA polymerisation instead of being added once the RNA is synthesised. This prevents the formation of 5'-triphosphate vRNA and limits the cellular anti-viral response, which will be described later [68]. In addition to their protective role against RNA degradation, some VPg can fulfill a second important role of the cap structure, promoting the vRNA translation initiation. This is the case of the *Caliciviridae* and *Potyviridae* 15 kDA VPg that is essential for vRNA translation initiation. This VPg directly interacts with eIF4E (the cap-binding protein) and the eIF3 complex (the 40S binding complex), which promotes the assembly of the translation initiation complex to the 5'-end of the vRNA (Fig. 9) [68, 72-75]. This allows VPg-vRNA to bypass the requirement for a direct eIF4E-cap interaction in order to initiate translation. This property is not conserved among all VPg, the *Picornaviridae* VPg is much smaller (2.5 kDA) and is not involved in the vRNA translation initiation [68]. These viruses instead rely on a highly structured RNA sequence called an internal ribosome entry site (IRES) to ensure their translation (this will also be described in more detail later on). All the (+)ssRNA viruses encoding a VPg benefit from its protective effect on the viral genome, but the *Caliciviridae* and *Potyviridae* VPg have evolved an additional function, promoting vRNA translation initiation. This VPs is a striking example of a cap substitute as it fulfills two critical functions of the cap structure, namely ensuring vRNA

**4.1. Viral proteins as substitutes for the cap structure**

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

stability and promoting translation initiation.

**Figure 9. Mechanisms of viral translation initiation**. The cap structure of RNA is specifically recognized by eIF4E, which recruits the cap-dependent translation initiation complex to the 5'-end of capped RNA. Alternatively, the viral VPg (covalently linked at the 5'-end of vRNA) can directly recruit eIF4E. The conserved viral RNA structure, located within the 5'-UTR, can directly promote translation initiation. These internal ribosome entry sites (IRES) are catego‐ rized into four different groups, each able to directly recruit a subset of initiation factors and the 40S ribosomal subu‐ nit in order to initiate translation. Of notice, some mechanisms of initiation require RNA scanning (left panel) by the initiation complex to reach the ORF while others position the ribosome directly adjacent to the transcription initiation site (right panel). Abbreviation : Human Immunodeficiency virus (HIV), West Nile virus (WNV), Encephalomyocarditis virus (ECMV), Hepatitis C virus (HCV) and Cricket paralysis virus (CPV), Open reading frame (ORF).

#### **4.2. Highly structured 5' RNA structure as an alternative to the cap structure**

The ribonucleic acid (RNA) is a macromolecule which, according to the central dogma of molecular biology, is a transient messenger carrying the genetic information required to pilot the protein synthesis. In addition to this canonical role, RNA, given its high chemical com‐ plexity, can fulfill additional roles including genome support, ordered three-dimensional structure and even catalytic activity [76]. Many viruses have exploited this capacity of RNA to form complex structure in order to promote viral replication. Some viruses, lacking enzymatic activity to synthesize or acquire a cap structure at the 5'-end of their vRNA, have instead selected a high-order structural RNA element upstream of their coding region. This peculiar RNA sequence can fold precisely and repeatedly into a definite three-dimensional structure. This ordered structure has numerous functions including binding to other macro‐ molecule partners. Those viruses use this *cis*-acting structure to bind directly or indirectly to ribosomal components in order to assemble the translation initiation complex at the beginning of their open reading frame (ORF). This promotes the cap-independent translation of viral genes. Such RNA structures bypassing the cap-dependency for translation initiation are called internal ribosome entry site (IRES). Many RNA virus families (e.g. *Dicistroviridae*, *Picornaviri‐ dae* and some *Flaviviridae*) use this structure to promote viral protein production. The diversity of viruses that have evolved distinct IRES structures can be divided into four categories that differ in their structure, length, mechanism of ribosome recruitment and robustness (Fig.9). The first group of IRES, which is the smallest and simplest, is encoded into the *Dicistroviri‐ dae* (e.g. Cricket paralysis virus) genome. This IRES consists of a 180 nt structure that is able to directly bind and recruit the 40S ribosomal subunit to the translation initiation site, and does not require any initiation factors nor methionyl-tRNA to initiate translation (Fig. 9) [77, 78]. The second group of IRES is similar to the first, but slightly larger with 330 nt. These include *Flaviviridae* of the *Hepacivirus* (e.g. Hepatitis C virus) and *Pestivirus* (e.g. Classical swine fever virus) genus. The second group of IRES is also able to directly bind the 40S ribosomal subunit, but requires the contribution of a limited number of initiation factors (eIF2 and eIF3) together with the methionyl-tRNA in order to initiate the vRNA translation [77-79]. Of notice, the RNA helicase eIF4A is not required for initiation of the group 1 or 2 IRES, an advantage that comes at the expense of a limited RNA unwinding capacity. Therefore the initial coding sequence of the ORF must be encoded by a non-structured RNA sequence, as an RNA structure will block translation initiation in the absence of helicase activity [78]. The *Picornaviridae* family viruses harbour IRES from the third and fourth groups and are similar in many regards. They are the largest IRES (450 nt) and the most complex. They do not directly bind the 40S ribosomal subunit and require canonical eIFs (eIF2, eIF3, eIF4A, eIF4B, eIF4G) together with additional proteins called IRES trans-activating factors (ITAFs) in order to recruit the ribosome and initiate translation [80]. The difference between these two groups lies in the positioning of the ribosome relative to the ORF. Group 3, found in the *Aphthovirus* (e.g. Foot-and-mouth disease virus) and *Cardiovirus* (e.g. Encephalomyocarditis virus) genera, recruits the ribosome at the initiating AUG codon. Group 4, found in the *Enterovirus* (e.g. poliovirus) and *Hepatovirus* (e.g. Hepatitis A virus) genera, recruits the ribosome upstream from the ORF and requires a scanning or shunting process to move along the RNA in order to reach the AUG codon and initiate translation [77, 78]. Of notice, those viral IRES (with the exception the of Hepatitis A virus IRES) are able to bypass the requirement for eIF4E, one of the limiting components of the capdependent translation initiation complex, to initiate their downstream ORF translation [81]. Encoding an IRES into the viral genome is an efficient mechanism evolved by viruses to fulfill a critical role of the cap structure, namely the translation initiation. The importance of this structure is exemplified by its remarkable degree of conservation. The case of the *Flaviviridae* family presents an interesting example: the members of the *Hepacivirus* and *Pestivirus* genera share a much closer homology between their IRES region than between their coding region, while members of the *Flavivirus* genus, do not have any IRES at all and synthesize a cap structure through a conventional viral RNA capping mechanism [78]. The emergence of viral alternatives to overcome the lack of a cap structure is a testimony to the crucial functions of this small structure for viral genome stability, replication and translation.

ribose methylations occurs via enzymatic activities located in the nucleus and cytoplasm, respectively [83, 84]. Similarly, many different viruses possess RNA 2'-O-methyltransferases in order to modify their mRNAs. The role of these methylations has however remained elusive until recently when it was demonstrated that 2'-O methylation of viral mRNAs enhances

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Viral infection normally results in the generation of immunological non-self RNA species. Pattern recognition receptors are a crucial component of innate immunity that are responsible for the detection of non-self RNAs [87]. Toll-like receptors (TLRs), retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide oligomerization domain (Nod)-like receptors (NLRs) are important pattern recognition receptors that recognize non-self nucleic acids of pathogens [88-90]. For instance, many TLRs can detect viral nucleic acids that are found in endosomes following the release of nucleic acids from infected cells [91-95]. This eventually leads to the activation of subsequent immune reactions. In contrast, RLRs detect viral nucleic acids in the cytoplasm of the infected cells during the early phase of viral replication [96, 97]. This detection leads to the induction of interferons and inflammatory cytokines which ultimately block viral replication and promote the activation of antigen-presenting cells in

RIG-I, MDA5, and LGP2 are important RLRs that can detect cytoplasmic viral RNAs and induce the expression of cytokines in order to establish a host antiviral state through the expression of numerous interferon-stimulated genes (ISGs) [98]. These include the protein kinase PKR and stress-inducible proteins such as IFIT1 and IFIT2 that can inhibit the protein synthesis machinery of the host cell [99-101]. What is the exact molecular signature found on viral RNAs that is detected by RLRs? Previous experiments demonstrated that RIG-I specifi‐ cally recognizes 5'-triphosphate groups that can be found on some viral RNAs [102-104]. Viruses must therefore hide or modify their RNA 5'-ends in order to evade the innate immune recognition through the addition of an RNA cap structure or through the addition of alternative 5' elements, such as viral proteins linked to the 5'end in order to hide their uncapped ends. This last strategy is used for instance by poliovirus which encodes a protein, VPg, which is covalently linked to the 5' end of the plus-strand genomic RNA [105]. Viruses that are unable to maturate their RNA 5'-end have instead evolved immune-evasion strategies to prevent ISGs induction. For instance, the Hepacivirus protease inhibits the signal transduction resulting

Recent studies suggest that 2'-O-methylation of viral RNAs can enhance the replication of viruses through evasion of the innate immune response [85, 86]. For instance, coronaviruses that lack a functional 2'-O-methyltransferase activity induce a higher expression level of type I interferon [86]. Moreover, these mutant viruses can replicate efficiently in the absence of some RLRs such as MDA5 [86]. Similarly, poxvirus and coronavirus mutants that lack 2'-O-meth‐ yltransferase activities show an enhanced sensitivity to IFIT proteins. Therefore, it appears that

virulence through evasion of intrinsic cellular defense mechanisms [85, 86].

**5.1. Innate immune response**

order to eliminate infected cells [98].

from RIG-I activation [106, 107].

**5.2. Importance of the RNA cap 2'-O-methylation**

#### **5. Recognition of the 5'-ends by the innate immune system**

In humans, the RNA cap structure harbors additional methylations at the 2'-*O* site of the first and second transcribed nucleotides of the mRNAs [82]. The addition of these supplementary ribose methylations occurs via enzymatic activities located in the nucleus and cytoplasm, respectively [83, 84]. Similarly, many different viruses possess RNA 2'-O-methyltransferases in order to modify their mRNAs. The role of these methylations has however remained elusive until recently when it was demonstrated that 2'-O methylation of viral mRNAs enhances virulence through evasion of intrinsic cellular defense mechanisms [85, 86].

#### **5.1. Innate immune response**

of viruses that have evolved distinct IRES structures can be divided into four categories that differ in their structure, length, mechanism of ribosome recruitment and robustness (Fig.9). The first group of IRES, which is the smallest and simplest, is encoded into the *Dicistroviri‐ dae* (e.g. Cricket paralysis virus) genome. This IRES consists of a 180 nt structure that is able to directly bind and recruit the 40S ribosomal subunit to the translation initiation site, and does not require any initiation factors nor methionyl-tRNA to initiate translation (Fig. 9) [77, 78]. The second group of IRES is similar to the first, but slightly larger with 330 nt. These include *Flaviviridae* of the *Hepacivirus* (e.g. Hepatitis C virus) and *Pestivirus* (e.g. Classical swine fever virus) genus. The second group of IRES is also able to directly bind the 40S ribosomal subunit, but requires the contribution of a limited number of initiation factors (eIF2 and eIF3) together with the methionyl-tRNA in order to initiate the vRNA translation [77-79]. Of notice, the RNA helicase eIF4A is not required for initiation of the group 1 or 2 IRES, an advantage that comes at the expense of a limited RNA unwinding capacity. Therefore the initial coding sequence of the ORF must be encoded by a non-structured RNA sequence, as an RNA structure will block translation initiation in the absence of helicase activity [78]. The *Picornaviridae* family viruses harbour IRES from the third and fourth groups and are similar in many regards. They are the largest IRES (450 nt) and the most complex. They do not directly bind the 40S ribosomal subunit and require canonical eIFs (eIF2, eIF3, eIF4A, eIF4B, eIF4G) together with additional proteins called IRES trans-activating factors (ITAFs) in order to recruit the ribosome and initiate translation [80]. The difference between these two groups lies in the positioning of the ribosome relative to the ORF. Group 3, found in the *Aphthovirus* (e.g. Foot-and-mouth disease virus) and *Cardiovirus* (e.g. Encephalomyocarditis virus) genera, recruits the ribosome at the initiating AUG codon. Group 4, found in the *Enterovirus* (e.g. poliovirus) and *Hepatovirus* (e.g. Hepatitis A virus) genera, recruits the ribosome upstream from the ORF and requires a scanning or shunting process to move along the RNA in order to reach the AUG codon and initiate translation [77, 78]. Of notice, those viral IRES (with the exception the of Hepatitis A virus IRES) are able to bypass the requirement for eIF4E, one of the limiting components of the capdependent translation initiation complex, to initiate their downstream ORF translation [81]. Encoding an IRES into the viral genome is an efficient mechanism evolved by viruses to fulfill a critical role of the cap structure, namely the translation initiation. The importance of this structure is exemplified by its remarkable degree of conservation. The case of the *Flaviviridae* family presents an interesting example: the members of the *Hepacivirus* and *Pestivirus* genera share a much closer homology between their IRES region than between their coding region, while members of the *Flavivirus* genus, do not have any IRES at all and synthesize a cap structure through a conventional viral RNA capping mechanism [78]. The emergence of viral alternatives to overcome the lack of a cap structure is a testimony to the crucial functions of

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

this small structure for viral genome stability, replication and translation.

**5. Recognition of the 5'-ends by the innate immune system**

In humans, the RNA cap structure harbors additional methylations at the 2'-*O* site of the first and second transcribed nucleotides of the mRNAs [82]. The addition of these supplementary

Viral infection normally results in the generation of immunological non-self RNA species. Pattern recognition receptors are a crucial component of innate immunity that are responsible for the detection of non-self RNAs [87]. Toll-like receptors (TLRs), retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide oligomerization domain (Nod)-like receptors (NLRs) are important pattern recognition receptors that recognize non-self nucleic acids of pathogens [88-90]. For instance, many TLRs can detect viral nucleic acids that are found in endosomes following the release of nucleic acids from infected cells [91-95]. This eventually leads to the activation of subsequent immune reactions. In contrast, RLRs detect viral nucleic acids in the cytoplasm of the infected cells during the early phase of viral replication [96, 97]. This detection leads to the induction of interferons and inflammatory cytokines which ultimately block viral replication and promote the activation of antigen-presenting cells in order to eliminate infected cells [98].

RIG-I, MDA5, and LGP2 are important RLRs that can detect cytoplasmic viral RNAs and induce the expression of cytokines in order to establish a host antiviral state through the expression of numerous interferon-stimulated genes (ISGs) [98]. These include the protein kinase PKR and stress-inducible proteins such as IFIT1 and IFIT2 that can inhibit the protein synthesis machinery of the host cell [99-101]. What is the exact molecular signature found on viral RNAs that is detected by RLRs? Previous experiments demonstrated that RIG-I specifi‐ cally recognizes 5'-triphosphate groups that can be found on some viral RNAs [102-104]. Viruses must therefore hide or modify their RNA 5'-ends in order to evade the innate immune recognition through the addition of an RNA cap structure or through the addition of alternative 5' elements, such as viral proteins linked to the 5'end in order to hide their uncapped ends. This last strategy is used for instance by poliovirus which encodes a protein, VPg, which is covalently linked to the 5' end of the plus-strand genomic RNA [105]. Viruses that are unable to maturate their RNA 5'-end have instead evolved immune-evasion strategies to prevent ISGs induction. For instance, the Hepacivirus protease inhibits the signal transduction resulting from RIG-I activation [106, 107].

#### **5.2. Importance of the RNA cap 2'-O-methylation**

Recent studies suggest that 2'-O-methylation of viral RNAs can enhance the replication of viruses through evasion of the innate immune response [85, 86]. For instance, coronaviruses that lack a functional 2'-O-methyltransferase activity induce a higher expression level of type I interferon [86]. Moreover, these mutant viruses can replicate efficiently in the absence of some RLRs such as MDA5 [86]. Similarly, poxvirus and coronavirus mutants that lack 2'-O-meth‐ yltransferase activities show an enhanced sensitivity to IFIT proteins. Therefore, it appears that 2'-O-methylation of cellular mRNAs has evolved as a molecular signature in order to distin‐ guish between self and non-self RNA during viral infection, and that ribose 2′-O-methylation in the cap structure of viral RNAs plays an important role in viral escape from innate immune recognition. Not surprisingly, it has been suggested that the development of pharmacological strategies that could inhibit viral 2'-O-methyltransferases could represent a novel therapy against viruses that replicate in the cytoplasm of infected cells [85]. In fact, it was previously shown that mutations of the 2'-O-methyltransferase catalytic residues can block or attenuate replication [22, 32] and that viral inhibitors such as sinefungin can inhibit methylation and suppress the replication of certain viruses, such as West Nile virus, in cell culture [108].

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