The enzymes for genome size increase and maintenance of large (+)RNA viruses François Ferron, 1,2 Bhawna Sama, 1 Etienne Decroly, 1 and Bruno Canard 1,2, * With sizes <50 kb, viral RNA genomes are at the crossroads of genetic, biophysical, and biochemical stability in their host cell. Here, we analyze the enzymatic assets accompanying large RNA genome viruses, mostly based on recent scientific advances in Coronaviridae. We argue that, in addition to the presence of an RNA exonuclease (ExoN), two markers for the large size of viral RNA genomes are (i) the presence of one or more RNA methyltransferases (MTases) and (ii) a specific architecture of the RNA-dependent RNA polymerase active site. We propose that RNA genome expansion and maintenance are driven by an evolutionary ménage-à-trois made of fast and processive RNA polymerases, RNA repair ExoNs, and RNA MTases that relates to the transition between RNA-to DNAbased life.

The acquisition of enzymes for genome size expansion and stability

Viruses survive and adapt to environmental changes while maintaining major functional and structural elements [START_REF] Agol | Emergency services of viral RNAs: repair and remodeling[END_REF]. This inner strength relies on an evolutionary paradigm involving seemingly antagonistic properties of genome stability and plasticity simultaneously. In a changing environment, viral loss of fitness (see Glossary) is buffered by genome evolution involving nucleic acid sequence adaptation (mutations, insertions, and deletions) and rearrangements (RNA recombination, gene acquisition/loss). In RNA viruses, this empirical exploration along fitness landscapes remains constrained by physical properties of the RNA molecule. To allow expansion and overcome the RNA biophysical limitation as a genetic material, physical protection, sequence stability, and specialized enzymes are required.

In light of recently discovered large (+)RNA virus genomes, we argue that they have achieved genome stability and maintenance through the acquisition and synergy of high-fidelity RNAdependent RNA polymerase (RdRp) and/or an RNA repair exonuclease (ExoN) and RNA methyltransferases (MTases), respectively. Thus, this opinion article focuses on these enzymes and their signature sequence accompanying large (+)RNA viruses.

Prebiotic RNAs and their evolution toward RNA and DNA genomes It is generally accepted that RNA appeared before DNA during evolution, supporting the existence of an ancestral RNA world [START_REF] Cech | The RNA worlds in context[END_REF]; together with primary peptides, short oligoribonucleotides acquired catalytic activity (ribozymes, ribonucleoproteins, and ribosome-like factories) and later peptide-coding capacity stored in their nucleic acid sequence. The appearance of membranes provided these primordial genomes (or 'replicators') new tools adapted to thrive in their environment. The RNA replication machinery of these replicators, or 'proto-RNA viruses,' arose from the primordial pool of genetic elements, while capsid proteins were captured from hosts and appeared later at different stages of evolution [START_REF] Krupovic | Origin of viruses: primordial replicators recruiting capsids from hosts[END_REF]. sizes range from a few kilobases (kb) for DNA viruses to multimegabases for higher-order eukaryotic organisms. The 'virus early' hypothesis [START_REF] Krupovic | Origin of viruses: primordial replicators recruiting capsids from hosts[END_REF] is conceptually compatible with this scenario: after this transition, proto-RNA viruses might have acquired and evolved their own polymerase, the RdRp, in parallel to the appearance of reverse transcriptase (RT), one of the most ancient DNA polymerase folds known. This is supported by the fact that DNA and RNA polymerases share a double psi-beta barrel fold, which is as ancient as the RNA recognition motif-containing fold or the palm domain fold found in viral RdRp and RT [START_REF] Koonin | The replication machinery of LUCA: common origin of DNA replication and transcription[END_REF]. Following this event that enabled RNA genome replication, tracking individual RNA genome size expansion and shrinkage at the level of individual species becomes a daunting task. Nevertheless, RNA viruses evolved together with their hosts through a myriad of events and mechanisms [START_REF] Wolf | Origins and evolution of the global RNA virome[END_REF].

In the tree of life, RNA viruses are the only group of organisms known to have an RNA genome.

RNA genome sizes range from a few kb up to ~41 kb in length [START_REF] Lauber | The footprint of genome architecture in the largest genome expansion in RNA viruses[END_REF][START_REF] Saberi | A planarian nidovirus expands the limits of RNA genome size[END_REF] in the case of (+)RNA viruses (Figure 1A,B). In this article, 'RNA virus' will refers to (+)RNA viruses unless specified otherwise.

Are there enzyme-specific markers of large RNA genomes?

The occurrence and distribution of enzyme-encoding genes according to virus genome size has been proposed to be an evolutionary marker in these large RNA genomes lying at this genome size limit [START_REF] Lauber | The footprint of genome architecture in the largest genome expansion in RNA viruses[END_REF]; indeed, a positive correlation exists between RNA virus genome sizes and the presence of RNA helicase [START_REF] Koonin | Origins and evolution of viruses of eukaryotes: the ultimate modularity[END_REF] and RNA ExoN domains [START_REF] Lauber | The footprint of genome architecture in the largest genome expansion in RNA viruses[END_REF]. Although the presence of the helicase seems to be an ancestral acquisition dictated by a functional requirement for RNA viral genomes >6 kb in size [START_REF] Gorbalenya | Viral proteins containing the purine NTP-binding sequence pattern[END_REF], the ExoN gene was acquired through evolution to provide an RNA synthesis proofreading system [START_REF] Eckerle | High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants[END_REF][START_REF] Denison | Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity[END_REF][START_REF] Bouvet | RNA 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex[END_REF][START_REF] Ferron | Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA[END_REF] essential for large RNA genome stability (discussed later).

In some large Nidovirales, ExoN is bound to a processive replicative RdRp and corrects mismatched bases appearing during viral RNA synthesis [START_REF] Bouvet | RNA 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex[END_REF][START_REF] Ferron | Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA[END_REF]. Over the past decade, a view of the overall repair process has been refined in coronaviruses [START_REF] Subissi | One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities[END_REF][START_REF] Smith | Thinking outside the triangle: replication fidelity of the largest RNA viruses[END_REF]. Beyond these acquisitions, large genome nidoviruses and nidovirus-like [START_REF] Lauber | The footprint of genome architecture in the largest genome expansion in RNA viruses[END_REF][START_REF] Shi | Redefining the invertebrate RNA virosphere[END_REF] viruses reside at the upper boundary of the largest RNA genome sizes, and their members code for an unusually large number of RNA modification enzymes carrying functions matching that of small DNA viruses, such as DNA bacteriophages [START_REF] Egloff | The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world[END_REF][START_REF] Smith | Coronaviruses as DNA wannabes: a new model for the regulation of RNA virus replication fidelity[END_REF]. Thus, markers of genome expansion can be tracked by following the acquisition of new genes (proteins) directly involved in RNA 'housekeeping.'

Is RNA synthesis fidelity increasing with genome size?

Most RNA viruses code for their own RdRps, generally quoted as being of 'low fidelity.' In DNAbased organisms, the mutation rate (μ g ) stays rather constant (~0.003 mutations per genome per replication) for genomes differing by several orders of magnitude [START_REF] Drake | Rates of spontaneous mutation among RNA viruses[END_REF][START_REF] Drake | A constant rate of spontaneous mutation in DNA-based microbes[END_REF], and sometimes strikingly lower (~10 -9 to 10 -12 ), as reported in more recent studies [START_REF] Sung | Drift-barrier hypothesis and mutation-rate evolution[END_REF][START_REF] Ossowski | The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana[END_REF]. By contrast, a μ g has been measured between ~0.8 and 6.5 mutations per genome per replication for (+)RNA viruses devoid of RNA repair system [START_REF] Sanjuán | Viral mutation rates[END_REF][START_REF] Drake | Mutation rates among RNA viruses[END_REF] and of ~0.075 for coronaviruses [START_REF] Sanjuán | Viral mutation rates[END_REF]. Thus, for large RNA genomes such as the latter, fidelity is higher by about one order of magnitude.

Strikingly, the mutation rates per nucleotide per replication cycle (i.e., weighting with genome size) for small RNA genomes do exhibit a lower fidelity of replication [START_REF] Bradwell | Correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage Qβ[END_REF]. One can thus ask if structural markers of RNA fidelity synthesis can be detected in RdRp sequences and structures and if they correlate with genome size.

The maximum possible intrinsic fidelity of viral RdRps

Fitzsimmons and colleagues suggested that viruses could have high mutation rates because it is hard to be simultaneously fast and accurate while facilitating adaptation [START_REF] Fitzsimmons | A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus[END_REF]. Viral replication needs to be fast for survival purposes over being accurate, thus leading RNA viruses to be Processive: a nucleic acid polymerase is processive when it catalyzes nucleotide incorporation continuously without dissociation from its template.

Processivity is defined as a number, which represents the number of nucleotides added to a growing strand per encounter of the polymerase with its nucleic acid substrate.

RNA viruses: viruses that have RNA as a genetic material. This RNA can be double-stranded (ds), such as reoviruses, or single-stranded (ss).

subject to other trade-offs of evolutionary significance. Thus, the question of RdRp fidelity in relation to genome size began to be indirectly addressed recently with the highly processive severe acute respiratory syndrome coronavirus (SARS-CoV) main RdRp complex made of nonstructural protein 12 (nsp12; RdRp) and nsp7/nsp8 (processivity factor). This complex, surprisingly, was found to exhibit a low fidelity of RNA synthesis in in vitro enzyme assays relative to other known RdRps belonging to smaller genome viruses [START_REF] Eckerle | High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants[END_REF]. This counterintuitive finding can be resolved as follows.In large Nidovirales, the RdRp complex is able to co-opt an RNA repair ExoN (carried in coronavirus nsp14 [START_REF] Ferron | Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA[END_REF][START_REF] Subissi | One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities[END_REF]). The current view, demonstrated in CoV-infected cells [START_REF] Eckerle | High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants[END_REF][START_REF] Denison | Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity[END_REF][START_REF] Eckerle | Infidelity of SARS-CoV Nsp14exonuclease mutant virus replication is revealed by complete genome sequencing[END_REF] and using purified enzyme assays [START_REF] Ferron | Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA[END_REF][START_REF] Subissi | One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities[END_REF][START_REF] Bouvet | In vitro reconstitution of SARScoronavirus mRNA cap methylation[END_REF] This is a logical evolution to cope with synthesis of its large (~30 kb) RNA genome: the faster RNA synthesis rate, the lesser detection in a given time window by cellular innate immunity guardians.

Beyond this example, we argue that through analysis of sequence alignments and structurebased studies, markers of RdRp active site fidelity can be determined (Figure 2). At least two specific structural features are apparent in the RdRp's catalytic site that we argue corelate with genome size. These structural features are located in motif F and motif C. Small genome (+)RNA viruses, exemplified here by Picornaviridae and Flaviviridae, possess a long side-chain amino acid (E or Q) at the second amino acid position after the absolutely conserved first Lys of motif F. Arteriviridae have a Gln at this position, which is corelated with the presence of the large genome (+)RNA Nidovirales signature sequence SDD at motif C. We feel it is important to note that none of these viruses have an ExoN, and thus their RNA synthesis fidelity must be more strictly controlled at the nucleotide selection/insertion step than that of viruses having an ExoN. Upon increase in genome size, slightly below 20 kb in size (starting with CASV;

Figure 2A), a short chain amino acid Ala (or Ser) appears in motif F, with a correlation with the presence of SDD in motif C and the presence of ExoN. The presence of this motif F Ala is a marker of RNA synthesis infidelity, as its short side chain leaves a wider space for the base of the incoming nucleotide [START_REF] Gong | Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase[END_REF][START_REF] Peersen | A comprehensive superposition of viral polymerase structures[END_REF]. In SARS-CoV, and together with an additional hydrogen bond provided by the Ser of the SDD motif, its presence has been suggested to be involved in the unusual nucleotide polymerization rate and infidelity measured during RNA synthesis in the absence of ExoN [START_REF] Shannon | Rapid incorporation of favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis[END_REF]. It is thus expected that large flaviviruses, which do not have ExoN, have expanded their genome size together with GDD, without evolving an Ala in motif F so far (Figure 2A,B). We therefore propose that large, Flavi-like genomes represent, up to now, the maximum achievable RNA synthesis fidelity without the help of an ExoN-mediated proofreading system. In other words, without presuming what actually drove genome expansion, one structural molecular signature for fidelity in the RdRp is located in motif C (GDD). what is actually promoting genome size increase or simply assisting large genome size maintenance. In particular, the discovery of large Flavi/Pesti-like viruses has uncovered 27.7-kb RNA genomes devoid of any obvious ExoN signature sequence [START_REF] Matsumura | Complete genome sequence of the largest known flavi-like virus, Diaphorina citri flavi-like virus, a novel virus of the Asian citrus psyllid, Diaphorina citri[END_REF]. ExoN domains, however, are present in other viruses showing innate immunity suppression and indirectly connected to replication [e.g., arenavirus nucleoproteins (NPs) [START_REF] Hastie | Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3′ to 5′ exonuclease activity essential for immune suppression[END_REF][START_REF] Papageorgiou | Brothers in arms: structure, assembly and function of Arenaviridae nucleoprotein[END_REF]]. In this latter case, genomes are segmented, though, and much smaller than those of large nidoviruses; their RNAs thus may not withstand the same constraints as large unsegmented (+)RNA genomes. It is thus logical to propose that other factors promote genetic stability of large RNA genomes: genetic stability should match chemical stability.

Large RNA viruses first solved a chemical RNA stability issue Genetic stability cannot be achieved on a biophysically unstable genetic material: the longer an RNA molecule, the greater the probability that it would lose its coding functionalities. Bases could lose their coding capacity through modification or elimination, or, more simply, the RNA could be cleaved. Cleavage may happen through host-mediated enzymatic nucleolytic cleavage [START_REF] Hartmann | Nucleic acid immunity[END_REF] or internal, cis-mediated self-cleavage through ribozyme-like reactions [START_REF] Jimenez | Chemistry and biology of selfcleaving ribozymes[END_REF]. To overcome the poor RNA stability, acquiring enzymes enhancing the chemical RNA stability or protective proteins seems a logical evolutionary path. Accordingly, it is now well known that RNAs can carry >100 epitranscriptomic RNA modifications [START_REF] Helm | Detecting RNA modifications in the epitranscriptome: predict and validate[END_REF], and viral RNAs are being increasingly reported as carriers of these epitranscriptomic marks (reviewed in [START_REF] Netzband | Epitranscriptomic marks: emerging modulators of RNA virus gene expression[END_REF]).

The 2′-hydroxy group stands out as a stability provider: it is a nucleophile promoting nonenzymatic alkaline internal RNA hydrolysis through a chemical mechanism shared by all of the naturally occurring, small, self-cleaving RNAs, by ribonuclease A and other ribonucleases [START_REF] Fedor | Structure and function of the hairpin ribozyme[END_REF], and either its 2′-O-methylation or its disappearance (in DNA) significantly increases nucleic acid stability. Likewise, SAM-dependent RNA methyltransferases (RMTases) are ancient protein folds susceptible to having been present early in evolution concomitantly with the RNA world [START_REF] Rana | Reviving the RNA world: an insight into the appearance of RNA methyltransferases[END_REF]. Thus, RNA 2′-O-MTases are excellent candidates to be ancillary stability factors.

In modifying RNA, RMTases may dampen the nucleophilic potential of chemical groups present in nucleobases, ribose, and phosphate that could promote intra-or intermolecular RNA cleavage.

Large RNA genomes are generally associated with RNA MTases

The analysis of large viral RNA genomes [START_REF] Shi | Redefining the invertebrate RNA virosphere[END_REF][START_REF] Shi | Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and related viruses[END_REF], such as genomes of nidovirus (~12-41 kb), flavivirus (~8.8-13 kb), flavivirus-like (~16-27.7 kb), and some unclassified Riboviria resembling mamastroviruses (up to ~20 kb), indicates that above ~17 kb of contiguous RNA, all viral RNA genomes contain at least one detectable RNA MTase signature sequence (Figure 3). Currently known RNA genomes >27 kb always carry both one ExoN and at least one MTase signature sequence. Unlike flavivirus-like genomes, which are so far limited to <30 kb in size, acquiring both ExoN and RMTase is linked to nidovirus-like genome expansion and maintenance.

Viral RNA MTases are not solely involved in RNA capping Two main MTase activities (RNA-cap N7-guanine and 2′-hydroxyl methylations) carried by viral enzymes participate in viral RNA capping [START_REF] Decroly | Conventional and unconventional mechanisms for capping viral mRNA[END_REF]. However, among the three virus taxa mentioned earlier, two do not rely on conventional RNA capping: Mamastroviruses use a protein cap (VPg) at the 5′-end of viral RNA [45], pestiviruses ( Flaviviridae) have no cap at all but rather an internal ribosome entry site (IRES) whose structure promotes protection and efficient translation [START_REF] Martínez-Salas | New insights into internal ribosome entry site elements relevant for viral gene expression[END_REF], and the situation is far from being clear for Nidovirales regarding the presence of a canonical RNA cap. Furthermore, arterivirus members do not code for any RMTase nor capping enzyme, the presence and structure of an RNA cap has been demonstrated only for one virus of the S1.

Torovirus [START_REF] Van Vliet | Discontinuous and nondiscontinuous subgenomic RNA transcription in a nidovirus[END_REF], and an RNA cap has only been inferred in other members of Nidovirales RNAs due to the presence of RMTase sequences [START_REF] Decroly | Conventional and unconventional mechanisms for capping viral mRNA[END_REF].

In parallel, it is interesting to note that several recent reports have shown that viral 2′-O-MTase specificities are not limited to RNA caps. The Ebolavirus MTase domain of the L protein methylates internal adenosine residues in RNA [START_REF] Martin | The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure[END_REF]. Similar observations had also been reported for Zika and Dengue viruses [START_REF] Dong | 2′-O methylation of internal adenosine by flavivirus NS5 methyltransferase[END_REF]50] Or did the RNA-to-DNA transition occur in small protoviruses, thus suggesting that large genome RNA viruses represent an evolutionary dead end after which size increase was no longer possible?

The appearance of DNA from RNA or precursors requires two essential enzymes: the thymidine synthase (TS), and the ribonucleotide reductase (RR). The RR is a metalloenzyme proceeding through free radical chemistry and metal-sulfur clusters [START_REF] Nordlund | Ribonucleotide reductases[END_REF], the latter metal clusters being of unusual abundance in large Nidovirales genomes [START_REF] Snijder | Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage[END_REF]. [START_REF] Egloff | The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world[END_REF][START_REF] Smith | Coronaviruses as DNA wannabes: a new model for the regulation of RNA virus replication fidelity[END_REF]. The latter nsp9 has no homologue in the RNA virus world but is structurally similar to,for example, the Escherichia coli T7 and T4 bacteriophage SSB gene 2.5 and gene 32, respectively [START_REF] Egloff | The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world[END_REF].

Concluding remarks

Due to their possible connection to the primordial RNA world, (+)RNA viruses could be considered as a special evolutionary case in the virosphere. Evolutionary forces, however, may have dictated to large proto-RNA viruses other solutions than embracing the DNA world: For (-)RNA viruses, genome segmentation (arena-, bunya-, and orthomyxoviruses) could be one, preventing a putatively costly maintenance of stable long RNA stretches while also providing an evolutionary benefit through genome reassortment [START_REF] Ojosnegros | Viral genome segmentation can result from a trade-off between genetic content and particle stability[END_REF]; the use of NP protecting the constantly attacked genomic RNA could be another means to offer some protection. Be it related or not, large

Nidovirales are the only (+)RNA virus genomes encoding their own NP for genome encapsidation.

We argue that size expansion and maintenance of large replicating RNA molecules resembling (+)RNA viruses have been promoted through both evolving an accurate RdRp enhanced by ExoN and RNA MTase-mediated stabilization (see Outstanding questions). Only a concerted improvement of RNA stability and RNA synthesis fidelity through these three essential enzymes might have been responsible, at least in part, for genome expansion and maintenance (Figure 4). Future studies on large (+)RNA genomes and their enzymes represent an exciting set of challenges ahead to fully understand the astonishing genome plasticity and evolutionary capability of RNA viruses.

These studies will certainly serve as science-based control measures against pathogenic RNA viruses, such as predicting the pandemic potential of emerging viruses, vaccine design, and drug resistance. In the current context of the SARS-CoV-2 pandemic and the recent shining success of mRNA-based vaccines, it seems worth investigating RNA modification enzymes promoting RNA stability, even though they may originate from exotic, nonpathogenic RNA viruses.

  measurement of the adherence to Watson-Crick rules during nucleic acid synthesis. Fidelity is a property of the polymerase and is expressed as the frequency of deviation (i.e., misinsertion) from Watson-Crick base-pairing rules. It equals the mutation rate when expressed per nucleotide inserted per template site. It is often blurred by natural selection of resulting mutated genomes, which thus represent only a part of the generated genetic diversity. Fitness: for viruses, the capacity to produce infectious progeny in a given environment. The definition is extended to the adaptation to their surrounding environment to survive, thrive, and reproduce for passing on the genetic information to the next generations. Mutation rate: the frequency of appearance of nucleotide changes. It can be expressed per nucleotide per template site (i.e., fidelity, see earlier), or per nucleotide per replicated genome, or per nucleotide per replicated cell, or per cell passage. When the number of replicated genomes in a cell or organism is unknown, it is impossible to estimate how many viral genomes have disappeared as a result of counterselection in the cell host. Nsp14: a bifunctional enzyme carrying both an RNA MTase in its C-terminus and a 3′ to 5′ exonuclease domain (ExoN) in its N-terminus, which excises mispaired bases ('errors') occurring during RNA synthesis and thus restores fidelity to levels compatible with a required genetic stability. Nsp14 ExoN is activated by nsp10, and both are products of expression of the coronavirus Orf1ab, further processed into nsp1-nsp16.

  ssRNA viruses can have positive (+) sense ssRNA [abbreviated herein as (+) RNA viruses], whose functional mRNA directly translates into proteins (e.g., coronaviruses), as opposed to negative (-) sense ssRNA [reported as (-)RNA], which first produces mRNA and then translates into proteins (e.g., orthomyxoviruses). Unless specified otherwise, RNA viruses discussed here are (+)RNA viruses due to their immediate evolutionary connection of proto-RNA viruses (see 'The acquisition of enzymes for genome size expansion and stability' in the main text). SAM-dependent RNA methyltransferases (RMTases): enzymes that use S-adenosylmethionine (SAM) as the cofactor to methylate the RNA acceptor molecule, generating S-adenosyl-homocysteine (SAH) as a by-product.

Figure 1 .

 1 Figure 1. Large (+)RNA virus genomes sorted according to genome size, with their respective color-coded family or genus.(A) Nidovirales, Flaviviridae, and Flavi-like and some unclassified Riboviria having a genome >8000 nucleotides (nt) in length. Table S1. (B) Simplified view of (A), showing representative order, family, and genus. Created with BioRender.The abbreviated virus names used throughout the text and figures are given in TableS1in the

Figure 1 (

 1 Figure 1 (continued).

Figure 2 .

 2 Figure 2. Sequences and structural features comparison of (+)RNA virus RNA-dependent RNA polymerase (RdRp) active sites order by genome size. (A) Motifs F (orange) and C (brown) alignment of a selection of representative viral RdRp sequences sorted by genome length. The presence (+) or absence ( -) of an exonuclease (ExoN) signature sequence is indicated on the left. Genome size is indicated and sorted by decreasing order on the right side of the table. Virus names are abbreviated according to the nomenclature (see Table S1 in the supplementary information online), and they are boxed in color according to their viral family. The catalytic motif C, which comes downstream of motif F, is either GDD or SDD. In motif F, the conserved lysine and arginine bordering the motif de fine the two NTPinteracting residues. The overall length of the motif F loop is variable from one family to the other and is illustrated by dots. The presence of a glutamate (E, purple box) in motif F is mainly corelated to the glycine (orange box) in motif C, else replaced by alanine, serine, or glutamine when the serine (green box) is observed in motif C. Alanine, conserved in Coronaviridae, is a marker of infidelity (see main text). (B) Conserved motifs F (orange) and C (brown) in RdRp structures of (top to bottom): severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)nonstructural protein 12 (Protein Data Bank accession no. 7BV2), Zika virus (PDB accession no.5TFR), and poliovirus (PDB accession no. 3Ol9). The RNA and pyrophosphate position in Zika virus were deduced from superimposition with the poliovirus RdRp structure. In motif F, the CoV polymerase carries an alanine (A547) instead of a glutamate residue (E161), removing a highly conserved interaction that positions the motif F arginine for interactions with the NTP and pyrophosphate. The serine (S759) of motif C (SDD) is able to interact with 2 ′-OH ribose in position -1 of the primer (broken line). Abbreviations: ExoN, exonuclease; RdRp, RNA-dependent RNA polymerase.

Figure 3 .

 3 Figure 3. Polyproteins of the three representative taxa,sorted by size in amino acids.

Figure 4 .

 4 Figure 4. A diagram showing evolutionary forces at work through synergy of three enzymes [RNA-dependent RNA polymerase (RdRp), exonuclease, and RNA methyltransferase (MTase)] to promote genome size increase and maintenance in (+)RNA viruses. Red arrows represent three positive forces for successful genome expansion through evolution. The respective measurable enzyme properties are represented in the inner circles, together

  . It is thus tempting to speculate that these Nidovirales and Flavi-like MTase

	substrate specificities are much wider beyond that of	a viral RNA cap. They could indeed be
	methylating viral	RNA internally and provide increased stability. Also, because one could argue
	that not all	virus genomes of 10-11 kb in size encode an RMTase, we propose, using the same
	logic as for the helicase, that it provides a selective advantage, as demonstrated and mentioned
	earlier for Zika and Dengue viruses.
	A 'battle' of RNA MTases in the infected cell
	Recent reports mention host cellular MTases acting on the invading viral	RNA [40]. For many
	viruses, these epitranscriptomic marks on viral RNA were described to play key roles in regulating
	several viral functions, including gene expression. In cellular methylation, the N	6 -methyladenosine
	is an abundant RNA modification found in viral	RNAs (for review, see [51]). These modifications
	induce pleiotropic function, which is regulated during the virus replication steps. For example,
	in influenza, N	6 -adenosine MTases have been shown to regulate splicing and export of viral
	RNA,	genome packaging,	and positively or negatively impact viral	gene expression [52].	In
	flaviviruses and others (reviewed in [50]), N	6 -methyladenosine dampens viral	expression (antiviral
	effect). Conversely, during an HIV infection, FTSJ3, a cellular RNA 2′-O-MTase, is recruited by
	transactivation response element RNA-binding protein (TRBP) to methylate HIV RNA at 17 posi-
	tions [53]. These methylations seem to have a proviral effect because they have been reported to
	favor viral	escape from detection by MDA5 and, in turn, the secretion of type I interferon. Thus,
	cellular and viral	RNA MTases may well	compete for RNA methylation in order to establish their
	respective phenotypic outcomes.

RNA processing enzymes, LUCRA, and a possible bacteriophage connection Could genomes of large (+)RNA viruses share vestigial features with the LUCRA? And would there be any clues of the RNA-to-DNA transition remaining in the largest (+)RNA viral genomes?

Acknowledgments

We thank Professor Olve Peersen and Dr Ashleigh Shannon for their thoughtful input and discussion of viral RNA-dependent RNA polymerase active sites and the viral RNA replication/transcription process in general. This work was supported by the Fondation pour la Recherche Médicale (FRM; Aide aux Équipes 2019-2022) and the Fondation Mediterranée Infection (Infectiopole Sud).

Declaration of interests

The authors have no interests to declare.

Supplemental information

Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.tibs.2021.05.006.