Viruses are a vastly diverse group of infectious particles with many different structures, mechanisms of function and ingenious strategies of invading host organisms for their own proliferation. One of the key features that ties viruses together as an inclusive group, is the reliance on living cells for replication and propagation. On their own, viruses lack the cellular machinery necessary for many life-sustaining functions including protein translation and metabolism. Regardless of the organization of a viral genome or the type of nucleic acid, infection of a host cell and viral propagation is dependent on the transcription of viral mRNA and, in turn, the translation of viral proteins as well as genome replication. Because viruses are dependent on host cell machinery for most of these processes, they have driven an outstanding virus-host co-evolution. Viruses that rely on the replication machinery of the host cell become cell-cycle dependent in their own replication. Furthermore, just as viruses have evolved ways to hijack necessary cellular proteins, cells have evolved complex mechanisms for fighting infection by detection and degradation of foreign mRNA. In order for viral mRNA to utilize host cell machinery, begin translation and remain both stable and undetected in the cytoplasm, it must contain the post-translational modifications of a host cell mRNA including, but not limited to, a 5’ cap structure. By disguising viral mRNA with the same structural elements found in host mRNA, the cellular defense mechanism can be evaded and protein translation may occur. The significance of the cap structure can be seen through the diversity of cap-synthesis pathways across vastly different viral families that all lead to the formation of a ubiquitous RNA 5’-cap. The 5’→ 3’ direction of nucleotide triphosphate (NTP) polymerization during RNA synthesis creates a nascent mRNA molecule with a 5’-triphosphate moiety resulting from the initial NTP on the 5’-end. Through the processes involved in cap synthesis, the pppRNA structure is transformed into a basic, cap-0 RNA structure (m7GpppN). Further 2'-O-methylations of the first and second nucleotides of the RNA may occur.
In this chapter, a number of processes used by viruses to synthesize, acquire or mimic a 5’ cap are explored to highlight the similarities and differences in the enzymatic mechanisms that lead to the maturation of a 5’cap on viral RNA and its importance in viral genome replication within a host cell.
2. Description of the RNA cap structure
To understand the importance of an RNA cap structure for viruses, it is crucial to first understand why this structure is essential to their eukaryotic hosts. Prokaryotic RNA transcription and protein translation are coupled due to the spatial proximity between DNA and ribosomes. In eukaryotic cells however, newly synthesized RNA transcripts undergo several nuclear post-transcriptional modifications, known as RNA processing, before they are exported and translated in the cytoplasm. These eukaryotic pre-mRNA modifications include the addition of a cap structure at the 5’-end, the splicing out of introns, the editing of nucleobases and the addition of a poly(A) tail at the 3’-end. RNA capping is a co-transcriptional process that occurs when an RNA molecule is 20-30 nucleotides in length. The cap structure consists of a guanosine residue, harboring a methylation in the N-7 position, which is bound to the terminal 5’-end nucleotide with a peculiar 5’-5’ triphosphate bridge (Fig. 1). This inverted link between the two nucleotides prevents RNA degradation by 5’-3’ exonucleases. The second important feature of the cap structure is the presence of the methyl group on the guanosine, which confers a positive charge that plays an important role in its specific recognition by specialized proteins. The cap structure fulfills many roles which ultimately lead to mRNA translation. In the nucleus for instance, the cap structure of pre-mRNAs is recognized by the cap binding proteins (CBP20 and CBP80). This cap binding complex (CBC) protects mRNA from degradation and assists RNA transport from the nucleus to the cytoplasm. Once in the cytoplasm, ribosomes and translation factors must be recruited for translation of mRNAs into proteins. The eukaryotic translation initiation factor 4E (eIF4E) specifically binds to the RNA cap structure . This association is mediated through stacking interactions between two aromatic residues of the eIF4E protein; the mRNA binding is further stabilized by specific hydrogen bonds between the positive charge of the 7-methylguanosine and an acidic residue . Upon cap binding, eIF4E assembles with eIF4G (a scaffold protein) and eIF4A (an RNA helicase) into the eIF4F complex . The scaffolding protein eIF4G recruits the small 40S ribosomal subunit through the eIF3 complex . The translation initiation complex then scans the mRNA for the start codon before recruiting the larger subunit of the ribosome, and translation of the open reading frame (ORF) takes place . Taken together, the roles fulfilled by the RNA cap structure are crucial for RNA stability and translation. Because of this, many eukaryotic viruses require strategies, such as RNA cap synthesis, in order to protect, replicate and translate their genomes in eukaryotic hosts.
3. Conventional and unconventional 5’ RNA cap synthesis mechanism
3.1. Canonical cap synthesis by different viruses
The importance of the cap structure in eukaryote metabolism has resulted in an evolutionary pressure for viruses to adopt a similar cap structure. A series of enzymatic reactions is required to synthesize a cap structure at the 5’-end of RNA. The most pervasive enzymatic pathway, also termed “conventional capping”, consists of three sequential enzymatic activities that are required to generate a functional 7-methylguanosine 5’-5’-triphosphate bridged cap structure. As a result of the directional 5’ to 3’ polymerization of nucleotide triphosphates (NTP) during RNA synthesis, nascent RNA bear at their 5’-end a triphosphate moiety (originating from the initial NTP). This 5’-triphosphate end of the RNA is first converted into a 5’-diphosphate end by hydrolysis of the terminal phosphate, or γ-phosphate, by an RNA triphosphatase (RTPase). This is followed by a two-step reaction catalyzed by an RNA guanylyltransferase (GTase). The enzyme first specifically binds and hydrolyzes a GTP molecule to form a covalent enzyme-GMP intermediate, which then catalyzes the transfer of the GMP moiety onto the 5’-end of a diphosphorylated acceptor RNA (ppRNA) in the second step of GTase reaction. Lastly, an RNA (guanine-N-7)-methyltransferase (N7MTase) uses S-Adenosyl methionine (SAM) as a methyl group donor in order to methylate the guanosine residue of the cap structure at the N7 position. This sequence of enzymatic modifications yields the minimal RNA cap-0 structure (m7GpppN). Subsequent methylation of the 2’-hydroxyl group of the first few nucleotides of the RNA can be catalyzed by a (nucleoside-2’-O)-methyltransferase (2’OMTase) again using a SAM molecule as a methyl-donor (Fig. 2). Further methylations on the caps proximal nucleotides convert a cap-0 structure into a cap-1 (m7GpppNm) or cap-2 (m7GpppNmNm) structure.
The conventional RNA 5’ cap synthesis mechanism is used by a majority of viruses in order to acquire a cap structure. Most DNA viruses together with the RNA viruses from the
3.2. RNA triphosphatases
The RTPase activity is the first of the three enzymatic reactions required to synthesize a cap structure. The RTPase hydrolyzes the γ-β-phosphoanhydride bond at the 5’-end of an RNA to yield an RNA 5’-diphosphate and inorganic phosphate (Pi). Viruses have evolved a wide variety of enzyme structures and mechanisms of action to fulfill the RTPase activity, a greater diversity than is seen with any other enzymatic capping activity. RTPases are classified as either belonging to the metal-dependent family or the metal independent family based on their cofactor requirements. As indicated by its name, the first family requires a divalent cation cofactor for its activity. This metal requirement is usually satisfied by Mg2+, although Mn2+ is also able to support the RTPase activity . This family of enzymes also shares the ability to hydrolyze free NTPs, again in the presence of a metal cofactor [5, 6]. The lack of substrate specificity is speculated to be a result of the chemical similarity between an NTP and the RNA 5’-triphosphate end. The metal dependent RTPase family is further subdivided into three distinct structural groups, namely the triphosphate tunnel metalloenzyme (TTM), histidine triad-like (HIT-like) and helicase-like RTPase (Fig. 4).
The TTM enzymes are found in chlorella virus, poxviruses, baculoviruses, mimiviruses and lower eukaryotes. All TTM RTPases fold in a specific, characteristic structure. An assembly of eight antiparallel β-strands to form a tunnel scaffold surrounding the active site (Fig. 4). The interior of the tunnel is dominated by hydrophilic amino acid side chains oriented toward the center of the tunnel creating a network of interactions for the triphosphate moiety of the substrate . Glutamate residues, within this amino acid network are also responsible for the coordination of the crucial cation cofactor . The recognition of the RNA substrate, primarily through its triphosphate moiety, could explain the activity of the TTM RTPase against NTP substrates. Interestingly, this NTP hydrolysis is not supported by Mg2+, but is rather dependent on Mn2+ or Co2+ . The coordinated metal ion, in conjunction with basic lysine and arginine, activates the γ-phosphate and stabilizes the pentacoordinate phosphorane transition state. A glutamate serves as a general base catalyst to activate the nucleophilic water for the attack on the γ-phosphorus according to a one-step in-line mechanism . TTM RTPases have been acquired by large DNA viruses from their hosts . Interestingly, modern
The HIT-like RTPase is so far only represented by the NSP2 enzyme of rotaviruses (dsRNA virus). The name of this family is based on the structural resemblance between the NSP2 C-terminal domain (CTD) and the ubiquitous cellular histidine triad nucleotidyl hydrolases (HIT). The NSP2 protein associates into an octamer to form a doughnut-shaped quaternary structure (Fig. 4) [9, 10]. RNA binding grooves are found at the surface of the doughnut-shape while the active site is buried deep in an electro-positive cleft on each monomer. Despite structural similarity with HIT, NSP2 appears to be catalytically distinct. The catalytic histidine triad requires a Mg2+ cofactor to hydrolyze the γ-β-phosphoanhydride and form a covalent phosphate-histidine intermediate . The enzyme harbours similar catalytic rates toward both NTP and pppRNA substrates. Increased affinity for RNA, conferred by the RNA binding grooves, is speculated to stimulate RTPase activity over NTPase activity
The helicase-like RTPases are found in a variety of ss(+) RNA viruses of the
The second family of RTPases is the metal-independent group. Higher eukaryotic viruses that rely on capping apparatus of the cell use the host metal-independent RTPase. Moreover, baculovirus also expresses such a metal-independent RTPase. Two striking differences between this enzyme family and the metal-dependent family, are its cation-independent mechanism of action and its inability to hydrolyze free NTP . Metal-independent RTPases are members of the cysteine phosphatase superfamily, sharing their signature HCxxxxxR(S/T) P-loop motif located in a deep positively charged pocket. The catalytic cysteine is located at the bottom triphosphate binding cleft formed by the characteristic α/β-fold ternary structure (Fig. 4) [15, 16]. The catalytic cycle fits a two-step phosphoryl-transfer reaction. First, the pppRNA γ-phosphate is attacked by the catalytic cysteine to form a covalent protein-cysteinyl-S-phosphate intermediate which results in the release of the ppRNA product. Next, a water molecule attacks the phosphocysteine to expel the inorganic phosphate and regenerate the enzyme . The metal-independent RTPase presumably evolved from the cysteine phosphatase ubiquitously found in higher eukaryotes and was later acquired by
3.3. RNA guanylyltransferase
The second step of the capping sequence is the GTase activity. GTase catalyzes the rate-limiting transfer of a GMP moiety from a GTP substrate to an acceptor ppRNA to yield an unmethylated cap structure (GpppN). GTases are members of the covalent nucleotidyltransferases superfamily which also includes the ATP- and NAD+-dependent DNA ligases and the ATP-dependent RNA ligases . This superfamily’s ternary structure is composed of the N-terminal of the nucleotidyltransferase (NT) domain fused to an oligobinding fold (OB-fold) domain in the C-terminal. These flexible proteins are able to undergo large conformational changes during their catalytic cycle. GTases share highly conserved structures and motifs, of which the hallmark KxDG(I/L) motif is present in nearly all GTases . The catalytic cycle of the GTase is a complex two-step ping-pong reaction involving multiple conformational changes. First, a GTase in a conformation where the OB-fold domain is distant from the NT domain (open conformation) specifically binds a GTP molecule. This is followed by the closure of the OB-fold domain toward the NT domain (closed conformation) which is stabilized by interactions between the bound nucleotide and residues from both NT and OB fold domains. This conformational change also creates a Mg2+ cofactor binding site, thus the closed conformation represents the catalytically active form of the enzyme [19, 20]. Upon Mg2+ binding, the α-phosphate of the GTP is sandwiched between the catalytic lysine (form the KxDG) and the metal cofactor. Deprotonation of the lysine leads to the attack on the α-phosphate of the GTP to form a enzyme-(lysyl-N)-GMP intermediate (EpG), concomitant with the hydrolysis of a pyrophosphate molecule . Following the catalysis, interactions between the bound guanylate and the OB fold domain are disrupted, leading to the reopening of the enzyme and the release of pyrophosphate. The reopening of the guanylylated enzyme allows for accommodation of the ppRNA, which is likely followed by the closure of the OB-fold domain. Closing of the OB-fold domain returns the enzyme to its catalytically active form, which promotes the transfer of the GMP to the acceptor RNA. A final reopening allows for unmethylated capped RNA to be released and the apo-protein to be regenerated (Fig. 5) . The active sites of the GTase are highly conserved, potentially due to their fairly complex catalytic cycle. Most viruses encode GTases that are, with respect to the active site, nearly identical to their eukaryotic host GTase, favouring the hypothesis of ancestral viral acquisition of the host GTase.
While nearly all GTases are highly conserved, a few recently discovered viral GTases are different. Little is currently known about those atypical GTases lacking the catalytic KxDG motif. Some segmented dsRNA viruses of the
3.4. RNA methyltransferase
The third step of the RNA 5’-end cap synthesis is the methylation of the cap guanosine by a N7MTase. An N7MTase adds a methyl group to the guanine at the N7 position in order to convert the GpppN into a functional m7GpppN cap-0 structure. The conversion of S-Adenosyl methionine (SAM) into S-Adenosyl homocysteine (SAH) provides the methyl group. N7MTases are members of the large SAM-dependent MTase family, which shares a low sequence identity but a structurally conserved SAM binding core. This SAM binding pocket, composed of a seven-stranded β-sheet flanked by six α-helices, ensures specific and proper positioning of the SAM molecule, while other structural determinants provide specificity for a range of methyl acceptors [23, 24]. For the N7MTase, those structural determinants are a positively charged RNA-accommodating groove and a GpppN binding pocket that forms extensive electrostatic interactions with the cap guanine, thereby ensuring specificity . Despite a broad network of interactions with both substrates (GpppN and SAM), no direct contact is made between the N7MTase and their substrate reacting group: the guanine N7 nitrogen (methyl acceptor) and the SAM CH3 (methyl donor). The methyl transfer is instead mediated by a direct in-line nucleophilic attack of the SAM methyl moiety by the guanine N7 nitrogen. N7MTases are not directly implicated in the transition state stabilization, but are rather optimizing the proximity and the spatial orientation between both ligands reacting groups. In addition, a favourable electrostatic environment further stimulates the catalysis . The degree of conservation among N7MTases is very high and most viral and eukaryotic N7MTases only differ in their accessory domain. A rare exception is the poxvirus N7MTase, which appears to bind SAM in a slightly different conformation. Moreover, some poxviruses, such as vaccinia virus, have evolved a heterodimer N7MTase. The vaccinia virus N7MTase D1 for example relies on its association with the accessory protein D12 to be fully active . The degree of conservation among N7MTases points toward a common eukaryotic ancestor acquired by viruses.
Lastly, some viruses infecting higher eukaryotes, such as
3.5. Gene organization of viral capping enzymes
In order to support viral replication and fitness, both the catalytic activity of viral enzymes involved in RNA capping as well as their localization within the cell, are crucial. Viral capping enzymes required for RNA capping have to be recruited at the site of RNA synthesis. Recruitment of the capping enzyme can be mediated by protein-protein interactions with either the RNA polymerase or a scaffold protein. While recruitment of the three distinct enzymatic activities is required in order to synthesise a cap-0 structure, the available surface for protein interactions at the RNA synthesis site is limited. Viruses have evolved multiple solutions to overcome this problem including the fusion of multiple enzymatic activities to the same polypeptide as well as protein-protein interactions between two capping enzymes to form a hetero-multimer (Fig. 6). A good example of protein-protien interaction is seen in
Some viruses have even evolved a highly efficient capping enzyme, fusing together all three or four enzymatic functions required for cap synthesis into what can be described as an RNA-capping assembly line.
3.6. Unconventional 5’ RNA cap synthesis mechanism evolved by different viruses
The capacity to properly cap RNA confers a distinct advantage to many eukaryotic viruses. Consequently, the selective pressure to maintain this structure is high, which is reflected by the degree of conservation among the viral capping proteins. Interestingly, this selective pressure is not directed toward the capping proteins themselves (RTPase, GTase and N7MTase), but rather toward their final product, the cap structure. Because of this, many viruses have evolved diverse biosynthetic strategies, divergent from the canonical RTPase→GTase→N7MTase pathway, allowing them to synthesize or acquire the final cap structure. This cap structure is in every aspect identical to the canonically synthesized one; only the enzymatic pathway varies. Many viruses families include members that use an unconventional 5’ RNA cap synthesis pathway. As of today, three unconventional 5’ RNA cap synthesis mechanism have been described.
3.7. The m7GTP RNA capping pathway
The m7GTP RNA capping pathway, also termed the
Of all known eukaryotes and viruses, the m7GTP RNA capping pathway is only used by members of the (+)ssRNA viruses, which points toward a eukaryote-independent emergence of this unconventional cap synthesis mechanism. In addition, the conservation of this capping pathway throughout distantly related viruses harbouring a broad spectrum of hosts, ranging from plants to animals, suggests an evolution from a common (+)ssRNA virus ancestor.
3.8. The GDP RNA capping pathway
The GDP RNA capping pathway, also termed the
3.9. The RNA cap snatching
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
The incredible diversity of RNA capping pathways, protein folding and enzymatic mechanisms of action that have been evolved by viruses all lead to the synthesis of the same ubiquitous structure is a testimony to the importance of the cap structure for viral genome replication and global viral fitness.
4. Viral alternatives to cap structures
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
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 complexity, can fulfill additional roles including genome support, ordered three-dimensional structure and even catalytic activity . 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 macromolecule partners. Those viruses use this
5. Recognition of the 5'-ends by the innate immune system
In humans, the RNA cap structure harbors additional methylations at the 2'-
5.1. Innate immune response
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 . 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 .
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) . 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 specifically 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 . 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 . Moreover, these mutant viruses can replicate efficiently in the absence of some RLRs such as MDA5 . Similarly, poxvirus and coronavirus mutants that lack 2'-O-methyltransferase 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 distinguish 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 . 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 .
This chapter explored the viral diversity of enzymatic activities and mechanistic pathways converging to the maturation of the 5’ cap on viral RNA. The cap structure provides tremendous advantages to eukaryotic viruses in terms of vRNA stability, gene translation and immune evasion. Some viruses have evolved enzymatic mechanisms of action unknown to the eukaryotic domain in order to synthesize this critical structure. Other viruses have developed novel cap synthesis mechanisms that generate a 5’ cap structure chemically identical to their hosts, yet formed by an entirely new process. Finally, particular viruses have also evolved unique mechanisms to steal or mimic the host cap structure. In conclusion, the incredible diversity and conservation of the mechanisms evolved by viruses to synthesize, acquire or mimic the 5’ cap structure is a testimony to the importance of viral RNA capping for viral replication, fitness and infectivity.
M.B. is a 'Chercheur Boursier' Senior from the Fonds de Recherche en Santé du Québec and a member of the Centre de Recherche Clinique Étienne-Lebel.