Cytosine drives evolution of <scp>SARS‐CoV‐2</scp>

Abstract

In this article, we show, in the specific case of SARS-CoV-2, that the role of cytosine-based metabolites used as cell growth coordinators is central to understanding both innate antiviral immunity and the evolution of the virus. An outbreak of atypical pneumonia, first noticed in Wuhan, Hubei province, China (Huang et al., 2020), developed into the COVID-19 pandemic (Jee, 2020) that displayed alarming similarities to symptoms caused by other β-coronaviruses (β-CoVs), SARS (Turinici and Danchin, 2007) and MERS (Bermingham et al., 2012) in particular. To contain the epidemic, efforts have been placed on diagnostic (Udugama et al., 2020), epidemiology (Park et al., 2020), vaccines (Zhang et al., 2020) and exploration of the use of old drugs to act as anti-coronaviral drugs (Du and Chen, 2020). Yet, all these endeavours underline an anthropocentric point of view that does not take properly into account the biology of the virus. We propose here that understanding at the molecular level how the virus multiplies and evolves in the metabolic context of its host may uniquely provide us with out-of-the-box solutions to fight the disease. To be sure, it is critical and urgent to be able to try and anticipate the future of the organism as it adapts to Homo sapiens. The multiplication of β-CoVs requires unique interactions with the host cell. Upon internalization, the virus is stripped of its envelope and, mistaken for a messenger RNA, its positive sense RNA genome is immediately translated into an RNA-dependent RNA polymerase (replicase) and proteins that allow it to hijack relevant host functions. This involves an intricate series of events, beginning with replication of the virus into a complementary RNA template that serves to generate both new viral genomes and several individual transcripts of that template (Sawicki et al., 2007; Chen et al., 2020). As an immediate demand, the virus must access the pool of ribonucleoside triphosphates needed for the transcription of 50–100 copies of the replicated RNA strand produced at each multiplication cycle. This makes the viral sequence highly sensitive to the idiosyncrasies of nucleotide metabolism. We thus expect that the cell's nucleotide general makeup will shape virus evolution as it inevitably mutates when producing its large progeny. Coronaviruses have evolved a specific family of functions meant to overcome some of this limitation via a proofreading step coupled to the function of its RNA replicase (Sexton et al., 2016). Yet, mutations remain unavoidable and viruses, which generate a huge number of particles within a single patient, will progressively integrate the various types of selection pressure that each virus variant faces. Selection pressure via efficacy of transmission multiplied by number of replicates per cell and selection pressure via intracellular availability of essential precursors (nucleotides, lipids, amino acids, carbohydrates) create a variety of bottlenecks for viral evolution (Kutnjak et al., 2017; Arribas et al., 2018; Orton et al., 2020). A second key feature of β-CoVs is that they are enveloped. Furthermore, some proteins of the virion are glycosylated, which involves tapping into the cell's resources of UDP-sugars (Wellen and Thompson, 2012; Mayer et al., 2019). Besides this uracil metabolism-dependent protein-tagging feature, we focus here on the phospholipids of the membranes, which also derive from metabolites involving pyrimidines, specifically from CDP-containing liponucleotides (Kuo et al., 2016; Woods et al., 2016; Lee and Ridgway, 2020). Briefly, we note that virus proliferation consists of reproduction of molecular sets whose chemical composition diverges grossly from the cell's average mRNA composition, already impacted by that of average nucleotide availability (Traut, 1994; Fig. 1). We highlight here the role of cytosine-based metabolites—and consequently that of guanine-based nucleotides—as critical coordinators of the global cell metabolism. We also document its likely consequence on the evolution of the virus. By definition, metabolism encompasses all chemical reactions connecting a limited panel of molecules so as to convert nutrients into cells. As parasites, viruses—especially those with small genomes, such as RNA viruses—do not generally code for functions that result in the construction of metabolic pathways. As a consequence, the chemical constituents of viruses derive from precursors obtained from food through metabolic reaction steps, all but a few encoded in the host cell's genome. The question thus arises about the adequacy between the virus genome-encoded biocatalysts that perform all stages of its proliferation, on the one hand, and, on the other hand, the chemical composition of viruses as infectious vectors, whose total quantity is to be maximized for the virus to be successful. Here we (i) highlight the deviation of SARS-CoV-2 RNA chemical composition compared with that of its human host; (ii) formulate a hypothesis grounded on the canonical organization of cytosine metabolism as a way to coordinate non-homothetic growth of cells—i.e., the simultaneous growth of the cytoplasm (three dimensions), the membrane (two dimensions) and the genome (one dimension)—, and point out the emergence of the endogenous antinucleotide viperin as a cognate adaptive antiviral metabolite and (iii) predict evolutionary trends of CoV-2 for maximizing compositional fitness—which seem to show up in ongoing mutation survey of radiative evolution. Compositional biases human mRNA vs (+)SARS-CoV-2. The histogram of the compositional difference between Homo sapiens mRNAs and the SARS-CoV-2 genome shows a considerable counterselection for the presence of cytosine (and guanine) nucleotides. The figure plots the log2[v(i)/h(i)] ratio of the content in the virus over the content in human cytoplasmic mRNAs. A striking feature of SARS-CoV-2 is that its genome appears to be depleted of cytosine nucleotides, pointing out an avoidance of cytosine as a most salient feature of its build-up. Because replication of the virus depends on a complementary RNA template, this deficiency is also reflected in a relative deficiency of guanine nucleotides. Indeed the virus is composed, on average, of 30.2% A, 19.9% G, 32.4% U and only 17.6% C (see also Fig. 1), probably reflecting the coupling between synthesis of viral particles and the host cell's metabolic capacity. Another noteworthy feature of the RNA sequence is that purines and pyrimidines are stoichiometrically balanced in the SARS-CoV-2(+) strand. This is interesting because the sequence does not follow the still enigmatic Chargaff's second parity rule, which would predict an equivalent amount of A and U as well as G and C (Forsdyke and Mortimer, 2000), and indicates that the virus is subjected to a metabolic balance equilibrating purines and pyrimidines. This is consistent with a study that used Flux Balance Analysis (FBA) that reviewed nucleotide stoichiometric availability (stoichiometric constraints as illustrated by Palsson and co-workers, for example, Schilling et al., 1999), and suggested guanylate kinase as a critical bottleneck in the build-up of the viral genome during infection (https://csbnc.informatik.uni-tuebingen.de/index.php/s/jd8rNcBJsmigFkz). When we compared the distribution of the four ribonucleotides in the human messenger RNAs with that in SARS-CoV-2, it appeared that cytosine was the rarest nucleotide of (+) sense virus genomes (exceptions are discussed in the Perspectives section), in a proportion considerably lower than in human mRNAs (not to mention tRNAs, rRNAs). Cytosine deficiency was matched by guanine deficiency (Fig. 1), as expected from the fact that the propagation of the virus results from a replication process incorporating complementary nucleotides. This process is amplified in the form of 50–100 copies, in a highly unsymmetrical operation, but this does not affect the pressure on the final guanine content. To understand how viruses tap in the host's metabolic resources, allowing non-homothetic growth in a stable manner as cells multiply, it is critical to understand how the construction of the cell's building blocks is coordinated to allow matching the growth of its cytoplasm (three dimensions), with the growth of its envelope (two dimensions) and the growth of its genetic setup (one dimension). We propose here that the selection of a specific subset of intermediary metabolites took place in the course of evolution as a way to smooth out non-homothetic growth. Briefly, among a variety of alternatives, cytosine nucleotides ended up as the coordinating metabolites, tying up growth of the cell's genome and of its membrane to central metabolism (Fig. 2). To be sure, de novo synthesis of nucleotides allows direct production of all triphosphates, including cytidine triphosphate (CTP). Nevertheless, there might remain some persisting negative trend against C, because CTP derives from uridine triphosphate (UTP) in a step that both requires adenosine triphosphate (ATP) and a nitrogen source, which makes availability of the molecule highly sensitive to energy and nitrogen availability. However, this type of negative pressure would equally apply to synthesis of ATP, the most abundant nucleotide in the cell (Zhang et al., 2008). This suggests that the organization of anabolic processes is not suitable per se to modulate the stoichiometry of specific nucleoside triphosphates (NTPs) according to a well-defined pattern. Then, what about their degradation and salvage? Synthesis and salvage of pyrimidines. In green, the central pyrimidine pathway for biosynthesis. The specific pathway for cytosine derivatives is in red. The dotted arrows mark catabolism. Additional routes are shown with dashed lines: purple for lipid synthesis and green for protein glycosylation. In blue we have shown the key enzyme which allows the recycling of pyrimidines via uracil. The parallel pathway for cytosine has not been discovered in any organism to date. [Correction statement added on 08 May 2020 after first online publication: Figure 2 has been corrected in this version.] At some point in all life cycles, metabolites are damaged then repaired or degraded then recycled, either as a whole or as parts. In the purine nucleotide salvage pathway, the key enzyme is adenine phosphoribosyltransferase (Wilson et al., 1986). In the same way, guanine is salvaged via hypoxanthine–guanine phosphoribosyltransferase (Balendiran et al., 1999). In pyrimidine salvage, uracil is recycled using uracil phosphoribosyltransferase (Li et al., 2007; blue arrow in Fig. 2). Yet, contrary to expectation, this does not go at all this way with cytosine. Surprisingly, no cytosine phosphoribosyltransferase has been identified in any living organism. For some reason, natural selection avoided retaining a direct route for cytosine salvage, despite that it does not seem difficult to evolve enzyme variants which would catalyse the reaction. The cytosine recycling process begins with converting cytosine-containing metabolites to cytidine then uridine, or cytosine to uracil, which is mainly scavenged directly by uracil phosphoribosyltransferase. Everything goes as if salvage of cytosine nucleotides had to go through deamination of cytosine-containing metabolites to uracil-containing molecules, followed by neosynthesis of UTP- and ATP-dependent amidation to CTP by CTP synthase (PyrG). This unique step is so essential that the pyrG gene is conserved in parasites (Aurrecoechea et al., 2009) and even in the smallest streamlined genome of an autonomous synthetic construct (Hutchison et al., 2016). What's more, the unique features of cytosine metabolism do not end up there, as CTP synthase displays a very unusual architecture. It forms filaments, named cytoophidia, in all organisms where its organization has been explored (Li et al., 2018). Cytoophidia create a strict compartmentalization of the corresponding activity (Liu, 2010; Sun and Liu, 2019). This organization is linear and therefore satisfies the bottom-up level constraints of non-homothetic growth (i.e., 1-D growth is even more constrained than 2-D growth, which itself is more constrained than 3-D growth), placing CTP uniquely at the crossroad of global metabolic controls. Correlated with 2-D growth, the synthesis of membrane lipids generates yet a further critical involvement of CTP-dependent metabolism for controlling non-homothetic cell growth. As a matter of fact, the double layer of phospholipids forming most membranes derives from cytosine-based liponucleotides (the raison d'être of cytosine-specific nucleotides in this makeup has not been investigated previously, but control of non-homothetic growth makes it a brilliant choice). This involves a variety of pathways initiated by CDP-diglycerides or chemically related analogues (Chauhan et al., 2016; McMaster, 2018). While the membrane lipid composition differs in the three domains of life, the general organization of the cognate pathways is similar, using CTP-dependent enzymes not only to form the lipid bilayer of the membrane but also to control its shape via its curvature (Cornell and Ridgway, 2015). In a metabolic development important in the present context where innate immunity must play a central role (Di Conza and Ho, 2020), CTP-dependent phospholipid synthesis is also essential for the generation of the endoplasmic reticulum (ER; Lagace and Ridgway, 2013). Finally, as now can be expected because of the importance of cytosine-based metabolites, the need for a specific organization of pyrimidine metabolism is implemented in further specific structures. As a case in point documented in many animal cells, the enzymes required for the onset of the de novo synthesis of pyrimidines, carbamoyl-phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase, and dihydroorotase (DHOase) make a multifunctional structure, known as CAD. All three activities are supported by a single 243-kDa polypeptide that forms hexamers and higher oligomers (Del Caño-Ochoa and Ramón-Maiques, 2020). Remarkably, this multifunctional enzyme is sensitive to proteolysis by caspase during apoptosis, indicating that pyrimidine metabolism is involved in this critical process (Huang et al., 2002). Further in line with the metabolic pathways organization explored here in relation with viral infection, CAD is highly expressed in leukocytes, where it enables Toll-like receptor 8 expression in response to cytidine and single-stranded RNA (Furusho et al., 2019). It was also observed early on that an increase in cytoplasmic CTP accelerated the rate of phospholipids synthesis in poliomyelitis-infected cells (Choy et al., 1980). Yet another most significant feature of CAD, relevant to our hypotheses, is that its activity is modulated by a dedicated viral protein during enteroviral infection (Cheng et al., 2020). This should prompt further analysis of pyrimidine metabolism in relation with SARS-CoV-2 infection, in particular looking for virus-encoded functions involved in interference with cytosine metabolism, as we now document. The requirement of cytosine nucleotides for the virus' genome and envelope synthesis is inevitably connected to the striking limitation in synthesis of cytosine-based metabolites. The most straightforward consequence of this metabolic design is that there is a force that will keep driving the cytosine content of RNAs to lower values, unless opposing processes (and selection pressure leading to discard organisms with too low cytosine content, for example, because this would create intolerable biases in the amino acid composition of the proteins coded by these genomes) had the upper hand during evolution. Coronaviruses, and other positive-sense single-stranded RNA viruses, produce plus strands at a 50- to 100-fold excess of their minus-strand replicated template. A further consequence of the replication process, however unsymmetrical, is that any pressure on a given base availability—here C—would affect its complement—G in our case—as noted above (Fig. 1). The mutations ordained to occur as the virus evolves will therefore reflect both the physicochemical forces acting during replication (typically triggered by cytosine deamination and reactions involving reactive species, resulting in formation of 8-oxoguanine, for example) and the metabolic setup of the host. As discussed in the previous sections, we expect a general selection pressure operating on CTP and tending, in the long run, to decrease the C content of the RNA virus. This is certainly qualified, however, by direct selection pressure on the functions that drive virus replication and propagation and operate on the corresponding codons, hence proteins. This is particularly important for the proline residue, encoded by CCN codons and essential in the folding of key viral protein domains (Li et al., 2014). The presence of a four codon insertion in the spike protein of SARS-CoV-2 is a case in point (Li et al., 2020). The C at the second codon position is also required to allow introduction of threonine or alanine residues in the viral proteins, while the first position is required for histidine and glutamine coding. In this context, it seems relevant to note that, in the SARS-CoV-1 in 2003, one of the critical changes with respect to innocuous counterparts was a leucine to alanine change at the junction between domains S1 and S2 of the spike protein and that this required a U to C change (Song et al., 2005). Now comes an extraordinary feature that accounts for the way animals control RNA virus diseases via innate immunity. It happens that the unique role of cytosine as a coordinator of global metabolism has been exploited by natural selection to endow hosts with antiviral processes based on interference with the metabolic involvement of this nucleobase. At least three responses have evolved in animals to further prevent virus multiplication, building up an efficient innate antiviral immunity based on cytosine metabolism. Animals, man in particular but this extends even to oysters (Green et al., 2014), have recruited an AdoMet-dependent biosynthesis pathway to construct a mimic of CTP, 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP, Fig. 3), using an enzyme named viperin, for virus inhibitory protein, and 2010; et al., that to and et al., The antiviral role of viperin appears to a variety of as expected from interference with cytosine metabolism. As at the end of the previous an is CTP-dependent lipid metabolism and but it also appears that it directly or via of the nucleotide et al., 2020), also with viral transcription or acting as a replication et al., 2016; et al., 2018; et al., 2018). and is the natural of the innate antiviral metabolite produced by analogues are as antiviral but if their chemical to produce the molecule will be immediately and to a must therefore be when the nucleoside analogues used as The second antiviral process involving cytosine that viral infection in is of the cytosine of in the viral In it has been shown that antiviral innate immunity as an efficient to the positive-sense RNA C virus et al., 2013). This as a is known to a variety of small RNA molecules on the cytosine of present in specific role is in the of molecules to the et al., et al., 2017; et al., 2019). this extends to the viral RNA genome or to its or it as is not there is in that can when are as in the context of et al., This might also affect during SARS-CoV-2 but this has not been It may be relevant that the in and of the viral that are for replication of the virus contain at the junction and As a role of another unique involvement of in of the viral sequence by the antiviral protein et al., 2020), which specifically to viral RNA degradation to viral RNA et al., 2020). As a consequence of this antiviral are in the genomes of many RNA However, the context where have a role in this process is as the antiviral response is not to their in any straightforward manner et al., 2020). The of cytosine functions thus seems to be key to innate immunity RNA viral we reviewed the role of a unique role of metabolism as the coordinator of non-homothetic cell growth. tap into the cell's resources, and this setup of intermediary metabolism an intracellular chemical pressure that must the evolution of the genome sequence of RNA viruses, in particular Because the virus sequence is replicated into a template, any pressure on the content in cytosine will be on its guanine content, as a role of guanylate kinase as a for antiviral drugs has been identified by et al., as a unique step whose would SARS-CoV-2 biosynthesis host cell (https://csbnc.informatik.uni-tuebingen.de/index.php/s/jd8rNcBJsmigFkz). hypothesis is that the availability of CTP (and hence of cytosine-based is a driving force in the way RNA viruses evolve a new progeny. The during of antiviral immunity that are based on the presence of cytosine in viral genomes this view We note however that, while in the long this trend a of C residues should to it may have been used as a natural for the virus to innate at least during the first of evolution of the the very fact that the genomes progressively their in particular in allow to the and innate while C will also for to with the is probably of a because will still with the construction of the viral In the long however, a of C in the genome will the proteins it in proline and thus considerably the evolutionary of the on a with viruses, which operating by tapping into yet another feature critical to anticipate the evolution of RNA viruses is as selection is based on the of functions that are a to the future of any When a function is the control of nucleotide metabolism via cytosine nucleotides synthesis and is to positive This that we should for RNA viruses that have overcome this limitation and for RNA as the viruses, which have genomes in the et al., 2016). is no straightforward that they code for that with CTP metabolism. However, despite the limited of their genome one of that of the of their genome for functions to proteins of the immediately have been shown to with the host cell's in certainly connected to metabolism and 2013). An from the present hypothesis would to up to this This would considerably our of the way viruses We are well of the of the present to the small number of and of the present we that, in view of the urgent we are with the COVID-19 it is important to our while to pressure that must have considerable importance in the evolution of viruses and in the design or new The emergence of as an antiviral by evolution as an innate immunity shows us a to We should however take in the development of nucleoside analogues as The of such molecules, may from the fact that they directly the activity of a viral for by the activity of A arises when an is by metabolic enzymes of the host into a of viral such as a chemically of the CTP or ATP, for The by the replicase of the into the RNA of the virus will its proliferation, as This be by the of the replicated RNA we then of a As discussed this is one of the antiviral observed for the viperin this result from incorporating into the viral allowing with more than one canonical to the genetic of the virus while it as we have from the use of which requires its into a triphosphate to the proliferation of et al., 2019). In the same way, the of of is to the of and it is into its nucleoside triphosphate et al., Yet, in the viral these analogues will be to nucleoside and which will be by the host cell's metabolism. The analogues will be via the enzyme ribonucleoside into then triphosphates, and into the cell's genome (Fig. 2). This a where of these antiviral will to in the human or mutations leading to or all that be observed in the the course of does not in of As a consequence, we that be during the required for of against For example, the is with the highly of and Fig. as well as via their into its triphosphate should therefore be of the of of these such as et 2020), of their antiviral This from with of the and from critical of and is of the and is of and of An the role of in the evolution of SARS-CoV-2 appeared after the present was for publication: deficiency in SARS-CoV-2 and of host antiviral and

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