The emergence of cooperative quasi-species consortia (QS-C) thinking from the more accepted quasispecies equations of Manfred Eigen, provides a conceptual foundation from which concerted action of RNA agents can now be understood. As group membership becomes a basic criteria for the emergence of living systems, we also start to understand why the history and context of social RNA networks become crucial for survival and function. History and context of social RNA networks also lead to the emergence of a natural genetic code. Indeed, this QS-C thinking can also provide us with a transition point between the chemical world of RNA replicators and the living world of RNA agents that actively differentiate self from non-self and generate group identity with membership roles. Importantly the social force of a consortia to solve complex, multilevel problems also depend on using opposing and minority functions. The consortial action of social networks of RNA stem-loops subsequently lead to the evolution of cellular organisms representing a tree of life. The neo-darwinistic narrative of the last century always assumed that the variation process is the result of random replication errors (mutations). It remained a curious explanation to reconstruct the emergence of organismic complexity by selection of accumulated beneficial errors. In contrast to this we assume both the evolutionary relevant genetic variations are the result of natural genome editing by viruses and related genetic parasites and the transfer and integration of virus derived genetic content into cell based organisms by infection events. Such events were coopted and exapted by the host and integrated remaining viral defectives such as mobile genetic elements and non-coding RNAs served to reprogramm gene expression and epigenetic markings. This we term virolution, virus driven evolution. The application of virolution overall to issues regarding the origin and evolution of life can provide us with a very distinct and new perspective on how living organisms emerge.2.The involvement of a virus-like process in the origin of life seems illogical. How can a molecular genetic parasite of the host system (virus) be involved in the origin of the host, even if the host is a more simplified RNA based living system? Would this not require the pre-existence of the host? But the term ‘virus-like’ includes parasites of RNA (stem-loop) replicators, even those simple RNAs that must have been ancestral to any RNA based life forms 3.Virolution means virus as mediators of evolution and as competent agents to edit host code 4.A ‘virus first’ perspective for understanding human evolution will likely seem counter intuitive or even preposterous to many readers This process has been called ‘virolution’; virus mediated evolution 5.Why would viruses promote novelty via the formation of complex networks able to contribute to host phenotypes We have long focused on the modest genetic adaptations associated with neo-Darwinian variation and selection. But whenever a host genome becomes colonized by non-ancestral endogenos retroviruses and related elements that replicate via RNA, a quasi-species consortia mediated process again applies to modify existing RNA networks that provided identity (being necessary for control and immunity) superimposing new and often multiple uses of stem-loop RNAs that are now engaged in and generate new identity networks 6.The term virus has a broad and almost instinctive meaning to many people with respect to disease. It is however, worthwhile exploring a current definition of this term in order to employ it with greater consistency and precision A virus is a molecular genetic parasite.A virus must be competent in the instruction system of its host systemA virus must superimpose (edit) new instructions onto the host system Viral instructions must promote maintenance of the virus (i.e. self identity compatible) which includes directed replication needed for either maintenance and/or transmission.Virus instructions can also simply include compelling the host to ‘maintain’ the viral instruction set (persistence) and replicate it along with the host.Viral instructions must oppose (i.e. damage) competitive instruction sets .These viral instructions may subvert (colonize) and manipulate opposing or competing instruction sets so as to maintain a coherent viral instruction system.The simplicity of RNA virus instructions requires that they be a coherent consortia of diverse RNA instructions (QS-C).In addition to these defining characteristics present in quasi-species consortia we would propose that the original ‘viral’ instruction system were simple stem-loop RNA replicators, as proposed for the RNA world 7.There are other important problems involving definitions that should be mentioned. These include the terms networks and systems. The real problem, relates to attempts to mathematically define these terms so that calculation based approaches can be applied to them However, it does compel us to use the terms networks and systems in a less mathematical (but popular) way. The concept of network in particular will be important for our discussions as it will relate directly to vital group identity which will require the specification of network membership characteristics We will often consider the issue of group identity and group behavior as these are the proposed to provide the foundations of social interactions. Therefore we seek to define a network from the perspective of a consortia of RNA agents and apply the strategies of these diffuse transmissive agents to explain the creation of new networks and redirecting existing ones 8.Another main question is the evolution of complexity. Let us remind the fact, that cellular life represent rare islands in a sea of viruses. As a consequence every cell based life is under constant infection pressure from its beginning until death Addiction modules represent at least two competing genetic parasite consortia , which try to invade host genomes. The competition targets not solely each other, but host immune system. This may result both the competing genetic parasites and the host immune system, can together install persistence and remain as a counterbalancing module For example, in the case of a restriction/modification module, this means 52 restriction enzymes are counterbalanced by 52 modification enzymes. This indicates how complex addiction modules may be constructed Most importantly, this new information representing genetic novelty by a persistent integration of counterbalanced coded genetic elements is not the result of error replication, but a result of module-like linked genetic contents. This fundamental difference to error replication narratives proposes new nucleic acid sequence constructions by integration of larger content arrangements into a coherent syntax without destroying the already existing sequence content This is an unique social force, because several RNA stem loop consortia cooperate by competition together with the host immune systeme and change host genetic identity by complementing into a counterbalancing module. The new capabilities that derive from integrated addiction modules are absent in the same host species which are not object to such infection events. Both, the competing viral clouds and the host genetic identity reach a cooperative upgrade of genetic complexity and addictive dependency on such infection events. Contextual circumstances  may destabilize such addiction modules and counterbalance may get weak or even out of control, the destructive toxin element may become active again and harm or even kill host.9.The development of neo-Darwinian thinking in 1930's stems directly from the foundation that natural selection acting on variation (mostly from replication errors) in individuals selects for the survival of fittest type variant. Thus the variation in offspring originates from the direct ancestor to the selected individual This explanation still invokes a central role for individuals. What then results are various scenarios, such as kin selection, or arms race ideas involving a serial one-upmanship and linear process of selection. Any networks that emerge will then need to stem form this same serial process. The process is not prone to punctuated bursts in evolution nor is it particularly prone to rapid emergence of complexity or novelty. Also, any associative or group behavior that emerges, such as altruistic behavior, will similarly stem indirectly from individual survival, as described by various kin selection or game theory models. This view has been well accepted for numerous decades and many current evolutionary biologist no longer question its basic tenants. Some even like to think of this as laws of evolution. But this is a view that emerged well before we understood the broad and ancient prevalence of virus The term virosphere has been introduced to describe this vast cloud of genetic information 10.How does virolution affect host evolution? Can it be providing some core, essential function needed for life? And more fundamentally, does virolution operate with additional principles, such as consortial group identities (QS-C) that can colonize and transform host, which fundamentally promote networks and complexity? If so, can these principles help us better understand the origin of life.Such virolution is what promotes the creation of new ‘systems’, not serial selection from errors The QS-C has to incorporate a new viral derived identity onto the host 11.RNA viruses have long been recognized as distinct agents from their host cells in that they were the sole survivors of the RNA world that still used RNA as a genetic molecule It was from this perspective in the 1970's that Manfred Eigen developed the quasispecies equations to explain the quantitative behavior of RNA populations that were generated via errors of the master fittest individual type template 12.Current knowledge about the evolutionary origin of placenta organ in mammals clearly indicates natural genome editing by persistent retroviruses Such a large scale retroposon colonization would seem to pose a highly genotoxic situation for the human genome, an idea which seems supported by genomic analysis It is now quite clear that most of this ‘junk’ is transcribed and that 95% of the transcripts are from repeated sequences that were retrotransposed 13.In the ensuing several decades, many laboratory observations were made that indicated more complex collective behaviors for viral quasispecies then were predicted by the quasispecies equations They can compete with and exclude related populations.They have minority populations that are crucial for overall fitness They can display heterogeneity important for fitness that is not observed in the consensus type They can suppress their own replication through lethal defection They can be composed of members that can complement and interfere with replication of the collective and many of these features can be observed in clinical infections such as humans with hepatitis C virus Thus quasi species are collectives that have positive and negative interacting members that are bound together for a combined fitness that depends on diversity Such dynamic diversity allows a population of otherwise rather simple agents (such as HIV-1) to defeat a highly complex and evolved system of adaptive and innate immunity in their human host With this clarification, it should become apparent that all RNA replicators  will have high rates of diversity generation (not error). In contrast to the error replication narrative such high rates of diversity generation can be termed also as high levels of non-directed creative productivity reminding us that living agents in populations does not replicate mechanistically in a machine-like manner but may search also for innovative solutions for unexpected context In addition, all genetic entities that replicate via RNA will also be prone to QS-C social (collective) behaviors Because of this much extended capacity relative to DNA, RNA can be considered as more active agents, with group behaviors that make it able to function as an agent-based population to affect its own activity and survival 14.To evaluate the QS-C and infectious perspective on the RNA world hypothesis, we will apply and explore the RNA-agent concept introduced above to the role of stem-loop ribozymes in the origin of life. The main objective is to incorporate the historically absent QS-C and parasitic perspective (with its inherent feature for group fitness) into the process that creates social RNA networks out of prebiotic elements. We will not explore early chemical evolution that might have led to the emergence of RNA molecules but will instead assume RNA has come into existence and follow its features from this perspective. One immediate consequence of this perspective is that we will be focused on collective features of RNA populations and will thus evaluate the chemical consequence of ribozyme QS societies, not individual replicators.This foundation immediately creates a situation in which collectives of molecules with multiple behaviors will have the primary role in promoting the origin of life. It will also be important early on to consider how these systems maintain coherence (group identity), as this is an essential feature. Indeed, a basic and continuing theme will be that a core function of stem-loop RNAs is to provide molecular identity through all of evolution, including recent human evolution The idea is then that individual members of stem-loop RNA populations were collectively able to invade (ligate into) each other to form a more stable and capable (ribozyme active) consortia with emergent, transformative and unpredictable abilities. These collective would lead to the origin of various ribosome and other RNA groups within cellular organisms still linked to its stem-loop tRNA origin 15.On the origin of the RNA world, short RNA oligomers formed by chemical processes needed to become longer RNAs able to perform template based catalysis. It has been proposed that the initial chemical formation of hairpin-like RNAs (stem loops) could provide ribozyme activity following a ligation based modular evolution that would yield ribozyme auto catalysis 16.A good starting point for the accumulation of complexity seems to be hairpin ribozymes whose activity can be controlled by external effectors Of particular interest is their ability to self ligate as this could promote the emergence of social RNA networks with group identity 17.The perspective social interacting networks of quasi-species consortia allows us to consider the role of stem-loop RNAs in the origin of the RNA world in which the action of individual agents can cooperate and be combined into a more capable collective action of a population. Thus the origin of spontaneous cooperating networks of stem-loop RNA replicators can be understood from this perspective Basically, for a network to be coherent and able to act collectively, it must limit membership to promote coordination by communication This does not require that only positive (e.g. replication) interactions be supported. Negative interactions, including interference will also be needed. For example, highly efficient run-away replicons would overtake a QS collective and yield only one RNA type. Thus the QS would lose complementing functionality and would also consume all substrates if they were not regulated. This situation presents a problem in those habitats with limited substrates, likely a very common state. Therefore, some level of self regulation (negation) in the collective would promote the survival of the collective, especially if these RNAs could interact with the substrate in a regulatory  manner. That efficient replicators become susceptible to parasitic replicators would provide an inherently spontaneous process of self regulation. Yet the collective will still need to promote replication when it is favored. Accordingly, it becomes important for members of the collective to be subjected to both positive and negative self regulation via RNA-RNA interactions. However, here too there must be some limits to self regulation as the collective cannot tolerate overly active self regulating members that will extinguish the collective.Thus we see that being a successful member of a collective has many (and multiple) behaviors associated with it. On top of that, as a QS-C replicates, these features will drift with time in a dynamic manner. In this context we can see that a random RNA stem-loop or a stem-loop RNA from a different QS collective would likely not be coherent with the other members of a particular QS. A social QS network is generally rather specific for its members. Group selection has already started. Indeed, many experiments with RNA viruses infecting humans and animals have shown a particular QS will exclude other QS of the same virus which results into immune function. Additionally such society membership is also time dependent in that the serial passage of the same viral QS will usually result in subsequent QS that preclude prior individual members of the QS. This behavior has often been called a ‘Red Queen’ behavior, but such a classical neo-Darwinian view does not incorporate or acknowledge the issue of group membership 18.As previously proposed group membership can also be promoted by the combined action of toxic agents linked to antitoxic agents Membership is thus crucial for living networks to emerge. In examining the literature relevant to QS, the RNA world and RNA network formation, we can indeed find some experimental evidence that supports QS and the spontaneous emergence of RNA networks Yet the biggest problem of all such studies is that there are few assumptions regarding the basic importance of social network membership Network membership needs to be prominently considered if we are to understand the origin of the ribosome and the genetic code. The emergence of the genetic code is not solely a molecular biological invention but the result of social interacting RNA consortia which needed many code using agents 19.The perspective on social networking of quasi-species consortia (qs-c) provides us with the opportunity to coherently explain how RNA stem loop groups mediate evolutionary novelty and genetic variations. The presented contribution basically traces social stem-loop RNAs from providing replicators, marking group identity, to the invention of ribosomes and translation. The half a century outlasting quasi species theory with its individual fittest type selection narrative is now revisited into social networking of quasi species consortia that provide us with a model how communicative interacting RNA groups generate genetic novelty, transfer even complex genetic content to cellular host organisms via infectious genetic parasites and evolutionarily change their genetic identity by persistence. The error-replication narrative of the last century can be replaced now by current knowledge about creative productivity of quasi-species consortia. The crucial roles of quasi species consortia membership which includes also minority types elucidates how such RNA populations may compete and cooperate in parallel in an indefinite and unpredictible way. As main drivers of genetic novelty they represent virolution being the essential pathway of the evolution of the tree of life.


Efficient RNA capping by the SARS-CoV-2 methyltransferase requires metal ions and a unique four-residue insert. O-methyltransferases catalyze the last step in the capping of viral RNAs, which protects the RNAs from degradation and prevents them from triggering host defenses. Minasov et al. report structures of the SARS-CoV-2 methyltransferase, a heterodimeric complex of the enzyme nsp16 and its coactivator nsp10, in complex with a short, capped RNA , the methyl donor SAM, and divalent metal cations. The metal ions and a four-residue insert of nsp16 were important for precisely aligning the RNA substrate in the active site for efficient catalysis. This insert is present in coronavirus but not in mammalian methyltransferases, suggesting this site as a potential target for the design of coronavirus-specific methyltransferase inhibitors.Virally encoded 2′- O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2′-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2′-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2′- Last, to form the Cap-1-RNA, a methyl group is transferred from S-adenosylmethionine (SAM) to the 2′-OH of the first adenosine ribonucleotide. For coronaviruses, this last reaction is catalyzed by the 2′-O-methyltransferase (MTase), a heterodimeric complex of nsp16 with the activator nsp10   demonstrated that methyl transfer is activated by divalent cations. SARS-CoV nsp14 is activated by MnO-MTase activity requires divalent cations. We used a custom-synthesized Cap-0-RNA substrate composed of the N7-methylated guanosine (m7G) attached through a triphosphate bridge to a short RNA (AUUAAA), which matches the naturally occurring ribonucleotides at the 5′-end of SARS-CoV-2 mRNAs  was over 10 times higher than that with the Cap-0 analog (m7GpppA) (We launched this research by confirming that the SARS-CoV-2 2′-ubstrate . In cont at 3 mM . These dm7GpppA) .7GpppA does bind to nsp16-nsp10 (Kd = 6.6 ± 0.3 μM) with threefold higher affinity than to nsp16 alone (Kd = 28.0 ± 5.5 μM) . In contrast, m7GpppG bound only to the nsp16-nsp10 heterodimer but not to nsp16 alone (Kd = 20.0 ± 2.7 μM) (fig. S1D and table S1). We also determined the binding affinities for the methyl donor SAM and the product S-adenosylhomocysteine (SAH) for nsp16 and the nsp16-nsp10 heterodimer . Neither SAM nor SAH bound to nsp16 alone, but both SAM and SAH bound to the nsp16-nsp10 heterodimer with Kd values of 6.9 ± 1.3 and 13.0 ± 1.2 μM, respectively. These biochemical and ITC studies together, as well as work by others  measurements showed that, although it is a poor substrate for catalysis, m7GpppAUUAAA substrate in the presence of Mg2+ or Mn2+. Multiple datasets were collected, and, ultimately, the three with the highest resolution and the best data statistics were selected for further analysis (table S2). Crystal #1 [Protein Data Bank (PDB) 7JYY] was soaked with substrates in the presence of 5 mM MgCl2 for 1.5 hours. In this crystal, we observed Cap-0-RNA, SAM, and Mg2+ . The capped RNAs in crystals #1 and #3 included the m7G cap, the first three ribonucleotides, and the phosphate group of A4. Crystal #2 contained the m7G cap, the first four ribonucleotides, and the phosphate group of A5.To gain insight into how metal ions stimulate catalysis, we took a structural biology approach. Crystals of nsp16-nsp10 in complex with SAM from different crystallization conditions were soaked with the custom-synthesized mand Mg2+ . Crystal crystal . Crystal7GpppA in the HBS is stabilized by stacking of the m7G and A1 bases with Tyr6828 and Tyr6930 residues, respectively  residues for the conformation of the mRNA in the catalytic site. The best resolved and most complete electron density for capped RNA was observed in crystal #2. The position of Uractions . The pho Asp6873 . The pho Ser6831 . The sta2+ or Mn2+ occupying the same site with similar interactions . In the nsp16-nsp10 and VP39 structures, the N7-methyl groups are nestled in the HBS pocket . In contrast, in NS5 and hCMTr1, for which methylation at the N7 position of the G0 is not required for the 2′-O-MTase activity . Although the m7G positions do not overlap, nucleotides N1 and N2 in all these structures are closely matched, which is consistent with the conserved mechanism of action and the structure of the catalytic site . Structural alignment of nsp16 with NS5, VP39, and hCMTr1 revealed that two important residues, Asp6873 and Lys6874, are located in a four-residue loop that connects β1 and αA (Comparison of the RNA binding site and the capped RNA conformation from the SARS-CoV-2 nsp16-nsp10 structure (PDB 7L6R) and the crystal structures of 2′-tic site . In all  Lys6874 . The supp moiety . Alignme1 and αA . This fo1 and αA .6873 and Lys6874 within the loop [Δ(D6873-K6874)]. Overall, Mn2+ stimulated the methyl transferase activity of nsp16 better than did Mg2+ for both the wild-type and mutant nsp16 proteins  values for activity of the D6873A and D6873G mutants in the presence of Mn2+ were shifted higher than values obtained for the wild-type enzyme  mutant showed much lower activity compared to the D6873A and D6873G mutants and the wild-type enzyme. At enzyme concentrations as high as 2 μM, the activity of ∆(D6873-K6874) reached only 45 and 65% of that of the wild-type max activity in the presence of Mn2+ or Mg2+, respectively. The results of activity assays support our structural finding regarding the importance of the four-residue loop that is unique for coronavirus nsp16 MTase as well as a preference for stimulation of the methyl transfer reaction by Mn2+.To test the effect of this insert on nsp16 MTase activity, we prepared nsp16 proteins with D6873A or D6873G amino acid substitutions or with a deletion of Aspproteins . At a 60 of Mg2+ . The EC5e enzyme . These sO-methylation events are essential for RNA metabolism and protection from cell innate immunity responses , which is conserved in all coronaviruses is critical for catalysis. The absence of this insert in mammalian MTases makes this region a promising site for the design of selective RNA-like coronavirus-specific inhibitors.A key advance of this work was the use of a custom-synthesized substrate RNA with ribonucleotides matched specifically to the sequences of the SARS-CoV-2 mRNA 5′-ends compared to earlier studies that used poly(C) RNA , 150 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 5% glycerol] for 2 hours. SAM was added to a final concentration of 2 mM. Freshly purified preformed nsp16-nsp10 heterodimer was concentrated to 4.0 to 5.5 mg/ml and immediately set up for crystallization as 2-μl crystallization drops (1 μl protein:1 μl reservoir solution) in 96-well crystallization plates.Recombinant nsp16 and nsp10 proteins were purified from 7GpppAUUAAA) was obtained from Bio-Synthesis Inc. . Cap-0 analog (m7GpppA) was obtained from New England Biolabs . The MTase activity was measured using the MTase-Glo Methyltransferase bioluminescence assay (Promega)  were plotted as a histogram using GraphPad Prism v9.Custom-synthesized Cap-0-RNA  using the Mantis liquid handler. After 1 hour at 37°C, the reactions were coupled with the detection and developing solutions using Mantis liquid handler and incubated as described above. Luminescence was measured in a TECAN Infinite M1000. The blank was defined as containing all reagents except Cap-0-RNA, and this value was subtracted for each corresponding point. The values were then reported as the percentage of MTase activity compared to the wild type under the same conditions. Assays were conducted in triplicate, and the experiment was independently repeated (n = 6). EC50 and the standard error were calculated using four-parameter logistic curve in GraphPad Prism v9 at 95% confidence interval.For determination of EC7GpppA Cap analog or the m7GpppG Cap analog . All samples were dialyzed overnight in ITC buffer . The concentrations of SARS-CoV-2 nsp10 or nsp16 used in titration experiments of individual proteins were 200 M and 40 μM, respectively. The concentrations of all substrates used were 500 μM. For titration experiments of the SARS-CoV-2 nsp16-nsp10, the two proteins were mixed to the final concentrations 25 and 200 μM, respectively, and incubated for 30 min at room temperature. A titration of SARS-CoV-2 nsp10 into nsp16 was performed at the 8:1 ratio to ensure no enthalpy was detected for the complex formation alone. The SARS-CoV-2 nsp16-nsp10 was titrated with substrates SAH and SAM at 375 μM, and each Cap analog at 280 μM. Individual titration data were analyzed with MicroCal PEAQ-ITC Analysis Software using a single-site binding model and nonlinear curve fitting. Each experiment was performed in triplicate, and the resulting values and standard error in the fitted parameters for N, Kd, ΔH, ΔTS, and ΔG were obtained and are summarized in table S2.Binding affinity was determined using a MicroCal PEAQ-ITC system  at 25°C. The sample cell volume was 200 μl, and the total syringe volume was 40 μl. For each titration, the first injection was performed using 0.4 μl, which was then followed by 18 additional injections at 2 μl per injection. The first injection was considered a void and was automatically removed from data analysis. Each injection was spaced by 120 s after a 60-s initial delay. SARS-CoV-2 samples of nsp10, nsp16, and nsp16-nsp10 were individually loaded into the sample cell and then titrated with either SAH, SAM, m2 or 20 mM MnCl2 in respective reservoir solutions and soaked for different time periods from 5 min to 17 hours. Crystals were cryoprotected with 4 M sodium formate or 25% sucrose in the respective reservoir solution and flash frozen in liquid nitrogen for data collection.Diffraction quality crystals grown from different conditions were transferred into 10-μl drops containing 5 mM SAM, 0.2 mM Cap-0-RNA, and into 10-μl drops with 5 mM SAM, 0.2 mM Cap-0-RNA, and 5 mM MgCl2, and cryoprotected with 4 M sodium formate. Crystal #2 was grown from 0.1 M citric acid (pH 5.0), 0.8 M ammonium sulfate, and was soaked for 6 hours with 0.2 mM Cap-0-RNA, 5 mM SAM, and 20 mM manganese chloride. Crystal #3 was grown from 0.1 M Hepes (pH 7.5), 0.5 M magnesium formate and was soaked for 6 hours with 0.2 mM Cap-0-RNA and 5 mM SAM. Crystals #2 and #3 were cryoprotected with 25% sucrose in the respective reservoir solutions.Crystal #1 was grown from 0.1 M sodium citrate (pH 5.6), 1.0 M ammonium dihydrogen phosphate, soaked for 1.5 hours with 0.2 mM Cap-0-RNA, 5 mM SAM, 5 mM MgClMore than 30 crystals were screened, and 32 datasets were collected at the beam lines 21ID-D and 21ID-F of the Life Sciences–Collaborative Access Team (LS-CAT) at the Advanced Photon Source, Argonne National Laboratory. Images were indexed, integrated, and scaled using HKL-3000  group corrections, which were created by the TLS Motion Determination (TLSMD) server (http://molprobity.biochem.duke.edu/). The capped RNA has a 5′-5′ triphosphate linkage between m7G and A1 with noncanonical RNA backbone geometry, which resulted in low RNA backbone scores in MolProbity reports. All structures were deposited to Validated SARS-CoV-2–related structural models of potential drug targets (https://covid19.bioreproducibility.org/) and to the Protein Data Bank (www.rcsb.org/) with the assigned PDB 7JYY , 7L6R , and 7L6T . Flexible parts of the structure, alternative side-chain conformations, and partial water molecules were fit into electron density maps at a lower σ level, which resulted in overall high real-space R-value Z-score (RSRZ) scores for all structures. All figures with models of the structures were created in PyMOL open source V 2.1 , murine hepatitis virus , human coronavirus , human coronavirus 229E , infectious bronchitis virus , DENV nonstructural protein NS5 , Homo sapiens , and VACV VP39 . The multiple sequence alignment was performed using Clustal-O  and merged with the coordinates of the structure with PDB code 7JYY using ESPript 3.x (https://itol.embl.de).The PDB coordinates of SARS-CoV-2 nsp16 and nsp10 were analyzed using the FATCAT (


The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest. Human noroviruses (HuNV) are a significant cause of gastroenteritis and account for approximately one-fifth of acute gastroenteritis across all age groups . Recent MNV replicates in laboratory cell culture lines and retains a similar genomic layout to HuNV ,7. MNV pAs with many viral proteins, the VPg proteins of noroviruses are multi-functional with a range of activities critical to the lifecycle of the virus. Nuclear magnetic resonance (NMR) analysis has shown that MNV VPg consists of a structured helical core flanked by unstructured regions at each end of the protein  10]. Du. Du10]. Picornaviridae and Potyviridae families, among others. The VPg proteins in these families share some similarities, including nucleotidylylation, however, additional functions and sequences of the proteins vary [VPg proteins are not unique to caliciviruses and have been identified in viruses in the ins vary ,21. Non-ins vary ,23,24 buins vary , a charains vary . Recentlins vary ,27,28.We identified a region of MNV VPg within the disordered N-terminus as a putative RNA binding site. RNA electromobility shift assays (EMSA) showed that MNV VPg and HuNV VPg were able to directly bind to RNA in a non-specific manner and we show that the MNV VPg binds to RNA through a basic patch of charged amino acids within the disordered N-terminal region that is conserved in noroviruses. Finally, we show that perturbation of the N-terminal amino acids critical for RNA binding also affects the ability of VPg to induce a cell cycle arrest.VPg protein sequences for MNV and HuNV GII.4 strain Sydney 2012 were obtained from GenBank . The nucleic acid binding potential of MNV VPg was assessed using the DRNAPred server , PONDR VE. coli chloramphenicol acetyltransferase (CAT) (GenBank BBB37551) were synthesised by Genscript . All constructs were confirmed by sequencing. Genes were ligated into a baculovirus transfer vector after a p10 promoter and recombinant baculovirus was produced using the Flashback Ultra system . A solubility tag, spidroin NT* [6-Strep II tandem affinity tag, the spidroin NT* fusion partner, an enterokinase cleavage site, and a flexible GGSRS linker, followed by the VPg or CAT protein.Synthetic genes for MNV VPg, including a K5A/K7A/R10A triple mutant (TM), a mutant with all K and R residues in the first 12 amino acids of VPg modified to alanine (K2-12), HuNV GII.4 VPg and roin NT* ,37, was Trichoplusia ni cells were infected with recombinant baculovirus expressing VPg proteins or CAT at a multiplicity of infection of one and incubated at 27 °C at 125 RPM for three days. Cells were harvested by centrifugation and lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, and 1% triton X-100. The protein was purified on Streptactin XT superflow beads  and eluted with 50 mM Tris pH 8, 150 mM NaCl, 10% glycerol, and 50 mM Biotin. Protein concentration was determined by absorbance at 280 nm on a NanoDrop One  and eluted proteins were stored at −80 °C. Expression and purification of recombinant proteins was confirmed by SDS-PAGE gel with Coomassie blue G250 staining.For protein expression, suspension cultures of TM Transcription Clean-Up kit according to the manufacturer’s instructions . Labelled RNA probes were eluted in nuclease free water and stored at −80 °C.The RNA binding potential of VPg was screened against a series of plasmids containing 12 pentaprobes (PP) gifted by Professor Joel Mackay (Addgene plasmid # 83326–83337) . PurifieRNA probes, approximately 150–200 bp, were designed to represent the 5′ and 3′ ends of the positive and negative-sense MNV RNA. The constructs were designed to include prominent RNA secondary structure elements near the 5′ and 3′ termini of the genomic and anti-genomic RNA. DNA templates for RNA probes were isolated by PCR and cloned into a pUC57 vector. All primers included a T7 promoter sequence and EcoRV restriction enzyme site, in addition to EcoRI and BamHI sites for cloning of the PCR products into the pUC57 plasmid .The 5′pos probe corresponding to the 5′ end of the positive-sense RNA represents nucleotides 1–155 of the MNV-1 genome and includes the 5′UTR and three stem loop RNA structures present at the 5′ end of the ORF1 sequence. The 3′neg probe corresponds to the 3′ end of the negative-sense RNA and is the mirror of the 5′pos probe. The 3′pos probe corresponds to the 3′ end of the positive-sense RNA, representing nucleotides 7158–7392 of the MNV-1 genome. The 3′pos probe contains three prominent stem-loops, the 3′ UTR and a short poly(A) tail. The 5′neg probe corresponding to the 5′ end of the negative-sense RNA is the complement of the 3′pos. The recombinant plasmids carrying the terminal sequences were linearised with EcoRV and RNA produced as described for the pentaprobes.TM Transcription Clean-Up kit according to the manufacturer’s instructions (Ambion).Generation of messenger RNA transcripts for transfection of RAW-Blue cells was performed as described previously . BrieflyUnlabelled DNA for competition assays was either produced from a single-stranded DNA template (TATGCGGGCGAAACTTCTGGGAATAGTCCTGACAACCCCTATTGCGATCAGCTCTTTTGCTAGCTCG) or a complementary sequence was annealed to the single-stranded DNA to produce double-stranded DNA.2 and 10% glycerol). Pentaprobe RNA was heated to 95 °C for one minute and then immediately cooled on ice prior to use, viral RNA probes were not heat denatured prior to the addition of VPg. For RNA binding reactions, ~100 nM of ATTO680 labelled RNA was added to protein samples at the indicated concentrations. The reaction was incubated in RNA binding buffer at 4 °C for 30 min. For DNA competition assays, a 100-fold molar excess of unlabelled DNA probe was also added to the binding reaction. Binding reactions were loaded onto a 5% native TBE acrylamide gel (29:1 acrylamide: bis-acrylamide) and electrophoresed in 0.5× TBE buffer at 120 V for approximately 40 min at room temperature. Gels were imaged on an Odyssey Fc imager in the 700 nm channel.Purified VPg proteins were buffer exchanged into RNA binding buffer . Cells were incubated for 12 h and harvested in 70% ethanol, washed in PBS (Dulbecco’s A), the RNA degraded with 0.1 mg/mL RNase A, and DNA stained with 50 μg/mL propidium iodide. The percentage of cells in each phase of the cell cycle was measured by fluorescence-activated cell sorting and the phases of the cell cycle assigned using MODfit LT 5.0 software . Expression of VPg proteins was confirmed by western blot using the appropriate primary and secondary antibodies; polyclonal rabbit anti-MNV-1 VPg [Cell cycle analysis was performed as described previously ,19. BrieNV-1 VPg , goat anp values of ≤0.05 were considered statistically significant.Cell cycle data presents the mean and standard deviation of three independent experiments. Results were analysed by one-way ANOVA with a Dunnett’s post-test, and Analysis of the MNV VPg sequence for predicted interactions with RNA and DNA using DRNAPred indicates that MNV VPg has potential for RNA binding, despite the absence of canonical RNA binding motifs such as a zinc finger domain or RNA recognition motif. In particular, the first 12 amino acids have probabilities between 0.91–0.83 for an interaction with RNA, whereas no regions of MNV VPg were predicted to interact with DNA a. The VPBioinformatic analysis indicates that MNV VPg contains regions of disorder, consistent with NMR data and disorder predictions of other norovirus VPg proteins ,40. The As RNA binding by norovirus VPg proteins was not known, we sought to determine the RNA binding potential of MNV VPg and whether it was sequence specific. We used a series of RNA probes, termed pentaprobes , which wE. coli [Recombinant MNV VPg protein was expressed and purified with an N-terminal NT* fusion partner and the resulting protein termed NT*MNV VPg a. The NTE. coli . ProductThe expression and purity of recombinant proteins used for RNA binding experiments was confirmed by SDS-PAGE gel with Coomassie blue staining b. The inIn some instances, a second shift band was apparent, which could indicate higher order structures or binding of multiple VPg proteins to a single molecule of RNA. As a negative control protein, NT*CAT showed no RNA binding activity across all 12 pentaprobes when compared with the RNA only control, confirming that a protein-RNA binding event was not a consequence of the NT* fusion tag. Removal of NT* using enterokinase resulted in MNV VPg still binding to RNA, confirming that NT* did not cause the binding of VPg to pentaprobes . CleavagPredictive analyses of VPg indicated a low probability for DNA binding a. The spVPg is essential for replication of the viral RNA and is covalently linked to the 5′ end of the viral RNA genome ,43. RNA Due to the conservation in sequence and common functions shown for norovirus VPg proteins, we tested if RNA binding would be a conserved function of other norovirus VPg proteins. A recombinant baculovirus was generated to express GII.4 Sydney 2012 strain HuNV VPg with an N-terminal NT*fusion protein. Purity of NT*HuNV VPg was confirmed by SDS-PAGE gel a and theThe N-terminus of norovirus VPg contains a highly conserved epitope rich in positively charged lysine and arginine residues . PredictTwo MNV VPg constructs were designed to test the importance of positively charged amino acids within the conserved sequence region for binding to RNA. The first construct created mutations, to alanine, at residues K5, K7, and R10 of the conserved epitope of MNV VPg (MNV VPg TM) a. The seThe conserved N-terminal region of norovirus VPg proteins is involved in binding of NTPs, nucleotidylation, and cell cycle manipulation ,17,25. WCell cycle analysis showed that at 12 h post-transfection, MNV VPg induced an increase, to approximately 70% of cells in G0/G1 phase with a corresponding decrease in S phase cells. b,d. MNV We used EMSA gels to show that MNV VPg binds to RNA as predicted by bioinformatic analysis and consistent with observed phosphocellulose binding of this protein .T.ni cells. MNV VPg was capable of binding to RNA both with the NT* fusion or without  play a crucial role in the regulation of cell cycle progression . These p


Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)+ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5′ or 3′ splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A) Genetic information is stored in genomic DNA. In higher eukaryotes, immature pre-mRNA is transcribed from genomic DNA by RNA polymerase II and undergoes a series of processing events, including capping, splicing and polyadenylation. The cap is added at the 5′ terminus while the RNA polymerase II pauses. Then, the transcription elongation continues. Introns are sometimes removed concomitantly to elongation in order to generate spliced mRNA. The 3′ terminus becomes polyadenylated once the RNA polymerase II has reached it. Mature mRNAs translocate to the cytosol through the nuclear pore complex to be translated into proteins.It is important to recognize exons, introns and their boundaries, which are regulated by cis-regulatory elements ,2,3. TheSplicing requires five U small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6, and more than 240 proteins . These c2+ concentration-dependent manner, and affects the alternative splicing of exon 41 and 42 of the inositol triphosphate receptor type 1 (IP3R1) [Previously, it has been reported that calcium homeostasis endoplasmic reticulum protein (CHERP) is localized in the endoplasmic reticulum and the perinuclear region, and colocalizes with the inositol 1,4,5-trisphosphate receptor (IP3R) . CHERP i (IP3R1) . Recentl (IP3R1) . However+ RNAs in the nucleus. To evaluate the causes of this nuclear accumulation, we performed RNA sequencing (RNA-seq) analyses, which indicated that the depletion of CHERP induced a dramatic change in alternative splicing, in particular, resulting in an increased retention of introns in transcripts. The properties of the intron retention and other alternative splicing events as well as the cell fate were also analyzed.Here, we investigated the function of CHERP in the nucleus and found that CHERP depletion induced an accumulation of poly(A)+ RNA localization (+ RNAs clearly accumulated in the nucleus after the depletion of CHERP to a similar extent as the accumulation induced by silencing the U2 auxiliary factors 65-associated protein (UAP56) or the nuclear RNA export factor 1 (NXF1), which are both involved in mRNA export. These results suggested that CHERP was involved in mRNA processing or nuclear export.To determine CHERP localization, cellular fractionation was performed and analyzed by Western blot. CHERP was found in the nuclear fraction of U2OS cells a, promptlization . Poly(A)To elucidate the function of CHERP in mRNA nuclear metabolism, we identified proteins interacting with CHERP by immunoprecipitating CHERP from cells stably expressing FLAG-tagged CHERP with an anti-FLAG antibody, and analyzing the interaction partners by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experiments were performed twice . The pro+ RNAs accumulated in the nucleus of CHERP-depleted cells seemed to be linked to III events. Moreover, dividing retained intron events into III and DII allowed identification of the poly(A)+ RNA that accumulated.To investigate the characteristics of introns retained in response to CHERP depletion, we considered separately increased intron inclusions (III) and decreased intron inclusions (DII) and found 290 III and 389 DII cases for FDR < 0.05 a, respecTo determine which mechanisms were triggered by CHERP to alter splicing events, we investigated the properties of introns. In both III and DII, the 5′ and 3′ splice site scores of introns affected by CHERP depletion were weaker than those obtained for the reference genes a,b. The The number of altered exon inclusions (EI) and exon skipping (ES) in cassette exons regulated by CHERP were counted. We found 2945 cases of EI and 3102 instances of ES with FDR < 0.05 a, implicTo elucidate the specificity of CHERP-regulated cassette exons, we performed a correlation analysis between the splice site properties and CHERP-dependent or -independent genes. In EI and ES, the 5′ splice sites in introns upstream and downstream of targeted exons were not significantly different from those of reference genes, except for 5′ splice sites in introns upstream of ES a. By conThe GO analysis revealed an enrichment of cell cycle genes in transcripts with EI induced by CHERP depletion, and those with ES were enriched in cell cycle or division, implying that CHERP played a role in cell cycle progression and cell viability. To investigate the role of CHERP in cell function, the cytoplasmic mRNA transcriptome was analyzed in cells with or without CHERP depletion. CHERP depletion induced the increase of the expression of 3934 transcripts and a decreased expression of 2822 mRNAs a. To det+ RNAs accumulated in the nucleus of CHERP-depleted cells, suggesting that CHERP was involved in mRNA metabolism in the nucleus [Here, the function of CHERP in mRNA metabolism was investigated. RNA fluorescent in situ hybridization (RNA-FISH) analyses revealed that poly(A) nucleus ,20. We t nucleus ,22. Our  nucleus . De Maio nucleus . RBM17 a+ RNAs accumulated in the nucleus of CHERP-depleted cells and CHERP was associated with U2 snRNP components. This prompted us to investigate CHERP’s role in mRNA splicing using next generation sequencing (NGS) and rMATS. Among alternative splicing events, retained introns were the most frequently observed, which was consistent with our observation that CHERP depletion induced poly(A)+ RNA accumulation in the nucleus [Poly(A) nucleus ,23. Alth nucleus . This di nucleus  and impl nucleus . It has  nucleus , suggest nucleus ,27.Alternative splicing is, importantly, regulated by the sequences of 5′ and 3′ splicing sites and their combinations ,28,29,30The depletion of CHERP induced alternative splicing changes, in particular of factors promoting the entrance into the M phase, including BIRC5, AURKB, CENPA, and BRCA1, which results in the reduced cytoplasmic expression of these factors ,46,47. OTM -Oligo Hybridization Buffer, and cell culture dish and plate , Alexa Fluor 594-labeled oligo-d(T)45 probe , Lipofectamine 2000 , bovine serum albumin, and 3--2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT) , the chemiluminescent reagent , Rever Tra Ace®, KOD Fx Neo, THUNDERBIRD® SYBR qPCR Mix, and restriction enzymes , RQ1 DNase-Free DNase , restriction enzymes , PlusOneTM Silver Staining Kit Protein , polyvinylidene difluoride (PVDF) membrane , ECL substrate  and trypsin-ethylenediaminetetraacetic acid (EDTA), doxycycline, hygromycin, Sepasol® RNA I Super G, and other general reagents .The following commercial chemicals were used: Dulbecco’s Modified Eagle’s Medium (DMEM) containing high glucose, 4’, 6-diamidino-2-phenylindole (DAPI), and SuperSep™ Ace , Fetal bovine serum (FBS), ULTRAhyb The plasmid DNAs, siRNAs and oligo DNA primers used in this study are listed in The following commercial antibodies were used: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse polyclonal antibody (FUJIFILM Wako), anti-β-actin mouse monoclonal antibody, anti-FLAG M2 mouse monoclonal antibody, anti-CENPA rabbit polyclonal antibody (Sigma-Aldrich), anti-BIRC5 rabbit polyclonal antibody , anti-AURKB mouse monoclonal antibody , anti-BRCA1 rabbit polyclonal antibody , anti-rabbit goat antibody labeled with Alexa Fluor 594 (Thermo Fisher Scientific), anti-mouse, anti-rat, or anti-rabbit goat antibody conjugated with horseradish peroxidase (HRP) . The anti-CHERP rabbit polyclonal antibody was a gift from Dr. Masatoshi Maki. The anti-U2AF2 mouse monoclonal and rabbit polyclonal antibodies recognizing NXF1 were gifts from Dr. Robin Reed. The anti-UAP56 rat polyclonal antibody has been described previously .To generate inducible stable cell lines, wild-type human CHERP cDNA was inserted into pcDNA5 FLAG FRT/TO. Cloning sites of individual plasmid DNA are described in For minigene splicing assays, the target region was amplified from human genomic DNA by PCR using KOD Fx Neo according to the manufacturer’s instructions, and cloned into pcDNA5 3 × FLAG FRT/TO. Mutant minigenes were obtained by mutation PCR. Sequences were confirmed by sequencing.TM T-RExTM cells (Invitrogen) and U2OS stably expressing H2B-GFP were maintained in DMEM, supplemented with 10% FBS 37 °C under 5% CO2.U2OS cells, Hela cells, HEK293 Flp-InTo establish stable cell lines expressing FLAG-CHERP wild-type, CHERP expression vector, pcDNA5 FLAG FRT/TO, was transfected with Lipofectamine 2000 according to the manufacturer’s instructions. Successfully transfected cells were selected using 100 ng/mL hygromycin in DMEM containing 10% FBS. A colony was picked up and expanded in a 6-well plate. To induce the expression of FLAG-CHERP, 1 μg/mL of doxycycline was added to the culture medium for at least 24 h. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot. U2OS cells expressing H2B-GFP were described previously ,49. 5 cells/mL) were seeded and cultured on coverslips. After 24 h, cells were fixed with 4% formaldehyde in cultured medium. Then, cells were washed once with phosphate-buffered saline (PBS) and blocked with PBS containing 6% bovine serum albumin for 1 h. The coverslips were incubated with anti-CHERP antiserum in PBS containing 2% bovine serum albumin, washed with PBS three times, and incubated with goat anti-rabbit antibody labeled with Alexa Fluor 594. DNA was counterstained with DAPI.Cells (1 × 105 cells/mL) were seeded and cultured on coverslips. After 24 h, cells were transfected with 20 nM siRNAs using Lipofectamine 2000, according to the manufacturer’s instructions. After 48 h, cells were fixed with 10% formaldehyde and permeabilized with PBS containing 0.1% t-octylphenoxypolyethoxyethanol/TritonTM X-100. The buffer was replaced with 2 × saline-sodium citrate buffer (SSC). Then, cells were prehybridized with ULTRAhybTM-Oligo Hybridization Buffer and incubated with 20 pmol Alexa Fluor 594-labeled oligo-d(T)45 probe overnight. Cells were washed successively with 2 × SSC, 0.5 × SSC and 0.1 × SSC. Images were taken at random using a Zeiss Axioplan 2  with an OLYMPUS DP70 camera . Segmentation of the nuclei and the cytoplasm and quantification of poly(A)+ RNA signals were performed using the CellProfiler cell image analysis software (version 3.1.5) [RNA-FISH was performed as described previously ,50. Brien 3.1.5) .2, and 1% octylphenoxy poly(ethyleneoxy)ethanol/NonidetTM P-40 (NP-40) on ice for 5 min. After centrifugation at 10,000× g for 1 min, the supernatant represented the cytoplasmic fractions. The pellets represented the nuclear fractions. Whole-cell fractions were prepared by lysing the cells as described previously [For the preparation of cytoplasmic and nuclear fractions, cells were scraped, washed with PBS, and treated with a cell lysis buffer containing 20 mM tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 8.0), 200 mM NaCl, 1 mM MgCleviously .2, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT). The cell solutions were then incubated on ice for 10 min, vortexed for 5 s, and centrifuged at 10,000× g for 10 s. The supernatants were collected as cytoplasmic fractions. The pellets were resuspended in an equal volume of solution containing 20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF and 0.5 mM DTT. The nuclei were lysed for 20 min on ice and centrifuged at 10,000× g for 10 min. The resulting supernatants were the nuclear fractions. The protein content in each fraction was determined by the Bradford assay . Protein samples were mixed with a 4 × SDS buffer  and boiled for 2 min.Proteins were extracted from the cytoplasmic fraction and nuclear extraction as follows. Cells were centrifuged and the pellet was suspended very carefully in three times its volume of lysis solution containing 10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgClTM T-REx TM cells stably expressing FLAG-CHERP and immunoprecipitation was performed as described previously [TM X-100, 0.2 mM PMSF and 0.5 mM DTT. The proteins on the beads were recovered in SDS buffer containing 250 mM Tris-HCl, 1% SDS, 0.002% bromophenol blue and 40% glycerol. After boiling for 2 min, the eluates were transferred to a new tube and separated by SDS-PAGE.Protein nuclear extracts were prepared from HEK293 Flp-In eviously . BrieflyThe protein samples were mixed with 4× SDS buffer and boiled for 2 min. Protein separation was performed using SuperSep™ Ace, 5–20%, 17-well. Silver staining of SDS-PAGE gels was carried out with Silver Staining Kit Protein, in accordance with the manufacturer’s protocol. Pictures were captured using the image analyzer LAS-1000 .LC-MS/MS analyses were described previously . ResultsSamples were separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked with 5% skim milk and then incubated with a primary antibody overnight at 4 °C. After washing, membranes were incubated with a secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. After careful washing, the signals were detected using ECL substrate with LAS 4000 mini .® Ⅰ Super G, and then treated with RQ1 DNase-Free RNase to remove genomic DNA, according to the manufacturer’s instructions. Then, cDNAs were synthesized by reverse transcription using Rever Tra Ace®, according to the manufacturer’s instructions. PCRs were performed using KOD FX NEO and analyzed by electrophoresis using either 1% agarose gel with a Tris-acetate-EDTA (TAE) buffer, or 8% polyacrylamide gel with a Tris-borate-EDTA (TBE) buffer. PCR products were visualized by a FAS-IV gel imaging system . Amplified PCR products were confirmed by sequencing.Total or cytoplasmic RNAs were isolated from whole cells or the cytoplasmic fraction, respectively, with SepasolU2OS cells (30% confluent) were transfected with 20 nM EGFP or CHERP#2 siRNA for 48 h. Whole and cytoplasmic RNAs were prepared as described above. The RNA-seq library was prepared using Truseq Stranded mRNA Library Prep Kit .http://software.broadinstitute.org/software/igv/home, accessed on 25 January 2022). Gene ontology (GO) analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID: version 6.7) [RNA-seq analyses of two biological replicates for each group were performed using the NextSeq High Output sequencer (Illumina). Sequence reads were mapped to the hg19 human genome using STAR (version 2.5.2b) . Mapped ion 6.7) ,58. The ion 6.7) , and braion 6.7)  program.ion 6.7) . Motifs ion 6.7) . SequencqPCRs were performed with the Thermal Cycler Dice Real Time System (Takara BIO) using Thunderbird SYBR qPCR Mix. Each sample was prepared in triplicate. The mRNA amounts were calculated by threshold cycle (Ct) values.Cells were cotransfected with siRNAs and minigene reporter plasmid DNAs. Briefly, cells were seeded at 15–20% confluency per well in 6-well plates. The next day, cells were transfected with siRNAs using Lipofectamine 2000. After 48 h of incubation, cells were transfected with minigene reporter plasmid DNAs (750 ng plasmid DNAs per well) using Lipofectamine 2000. After one day of incubation, cells were collected for the preparation of total RNA, as described above. Expression of the minigene reporter was analyzed using RT-PCR, followed by electrophoresis.2 for 16 h.Cells expressing H2B-GFP were seeded on 35 mm-diameter glass bottom dishes  and transfected with siRNAs using Lipofectamine 2000, as reported previously . After s3 cells/mL in a 96-well plate and cultured for 24 h. siRNAs were introduced to each well and incubated for 48 h. Afterward, 5 μL of 5 mg/mL MTT was added to each well and incubated for 4 h. The cell culture plate was then centrifuged at 400× g for 5 min. The supernatant was removed and cells were solubilized with 10 mM NH4Cl containing 10% SDS (pH 7.0). Cell viability was estimated by measuring the optical density at 600 nm.Cell viability was assayed by colorimetric MTT assay ,63. U2OS


Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed. Picornaviridae including, foot-and-mouth disease virus (FMDV), human enterovirus 71, and hand, foot-and-mouth disease virus, are positive-sense single-stranded RNA viruses. The genomic RNA of approximately 8500 nucleotides is enclosed within a non-enveloped capsid with an icosahedral structure [pro and 3Cpro) into thirteen mature viral proteins [The members of tructure . In infeproteins . The P1 pol-mediated uridylylation of 3B [The 5′UTR of picornaviruses ranges from 415 to 1451 nucleotides, depending on viral species. It forms a complex secondary structure involved in viral replication and translation . FMDV 5′on of 3B , later ron of 3B . Thus faon of 3B .pol [pol binding [The 3′UTR also plays a role in the functional IRES element. It acts as a translation enhancer to mediate the initiation of cap-independent translation of several plants’ RNA viruses . Moreovepol . The dowpol  due to i binding . The 3′U binding . Additio binding .Reverse genetics of picornaviruses, including FMDV, mainly depends on in vitro transcription and RNA transfection ,18,19,20Herein, we have proposed the establishment of a plasmid DNA-based FMDV minigenome that can efficiently generate uncapped mRNAs within the cytoplasmic compartment. This imitates the generation of a viral genomic RNA-like molecule, which can be used as a template for the cellular translation process. In this study, our FMDV minigenome together with the helper plasmids could proficiently drive the transcription and translation of the green fluorescent protein (GFP) gene in Baby Hamster Kidney-21 (BHK-21) cells. We have also demonstrated the application of the FMDV minigenome for cell-based antiviral drug screening. In a parallel study, this minigenome carrying the full FMDV polyprotein coding region, when transfected with the two helper plasmids, could produce infectious FMDVs. We believe that not only is this system suitable for FMDV but it can also be applied for other picornavirus minigenomes. Moreover, it can be used for foreign protein expression such as a DNA vector but working solely in the cytoplasm.2. The FMDV used in this study was provided by the Bureau of Veterinary Biologics, DLD, Thailand, including serotype A; A/Lopburi/2012 (A/Lop), type O; O/TAI/189/1987 (O189), and type Asia1; Asia1/2013. All viruses were cultivated in BHK-21 cells, which were maintained in MEM supplemented with 2% FBS.BHK-21 cells (ATCC) were cultured in Minimum Essential Medium  supplemented with 10% fetal bovine serum (FBS) , 100 IU/mL ampicillin, and 2 mM L-glutamine and incubated at 37 °C with 5% CO®, Taipei, Taiwan) following the manufacturer’s protocol. cDNA was generated from the RNA template using Superscript III RT  according to the manufacturer’s instruction. Briefly, the reverse transcription (RT) reaction contained 200 ng of random hexamer primers, 1 µL of 10 mM dNTP, 1 µg of RNA, and dH2O up to 13 µL. The RT master mix consisting of 4 µL of 5× FS buffer, 1 µL of 0.1 mM DTT, 40 Units of RNase OUT, and 200 units of Superscript III RT was prepared before adding into the reaction tube. The reaction was incubated at 55 °C for 1 h prior to being stopped by incubation at 70 °C for 15 min.Viral RNA was extracted using a viral nucleic acid extraction kit  of 0.01. Three replicates were included for each virus. After an hour of virus adsorption, the inoculum was then removed, and the cells were washed once with MEM and maintained in 2 mL of MEM containing 2% FBS. The infected cells were incubated at 37 °C with 5%CO® University Edition . Once the analysis showed at least one mean with a statistically significant difference, the post-hoc comparison with the Tukey method was applied to demonstrate the different one. In addition, the difference of mean titers at each time point was also evaluated with the GLM and Tukey post-hoc comparison. The p-value less than 0.05 was considered as a statistical significance. The viral growth kinetics were plotted between the virus titers and hours post-inoculation (hpi). The procedures containing live viruses were performed at the Bureau of Veterinary Biologics, DLD, Thailand.The log10 of virus titers were analyzed for the difference between mean titers of all 6-time points of each isolate with the repeated measure ANOVA using the generalized linear model (GLM) method available in the SASXhoI and StuI recognition sequences.To generate the FMDV minigenome, the entire FMDV O189 UTR was PCR amplified, cloned, and sequenced. The 5′UTR fragment was divided into three overlapping DNA fragments—S-fragment, poly C tract, and the large fragment (l-fragment)—for amplification by fusion PCR. In the first reaction, the S-fragment, poly C, and l-fragment were amplified with each specific primer pairs  using th®, Piscataway, NJ, USA), which was subsequently cloned into the plasmid pUC57 vector , resulting in pUC57_3′UTR. The DNA cassette containing the sequence T7 promoter-pol I promoter-FMDV O189 5′ UTR in 5′ to 3′ direction was excised with NruI/StuI restriction enzymes  from the pKLS1 vector and then ligated into pUC57_3′UTR upstream the 3′UTR, to create pKLS3 vector (GFP) was amplified from pEGFP-N1  by using primers EGFP_F and EGFP_R  was modified from pKLS1. Briefly, the sequence of human polymerase I (Pol I) promoter was amplified using pDZ_NP2  as the t3 vector A. The end EGFP_R . The GFPKLS3_GFP B.®-T7 in vitro Transcription Systems  to produce an RNA molecule containing the Pol I-FMDV_5′UTR-GFP-FMDV_3′UTR-poly A sequence with the length of approximately 2.2 kb. Briefly, 500 ng of the plasmid was mixed with 4 µL of the 5× Transcription Optimized Buffer, 0.1 M DTT, 20 U of Recombinant RNasin® Ribonuclease Inhibitor, 2.5 mM of each rNTP, 15 U of T7 RNA Polymerase, and nuclease-free water to the final volume of 20 µL. The reaction was incubated at 37 °C for 1 h, and the RNA product was analyzed by agarose gel electrophoresis.The activity of the hdv ribozyme was examined by in vitro transcription. The closed-circular plasmid DNA, pKLS, was used as the template for in vitro transcription with the application of the RiboprobeE. coli BL21 (DE3) . The T7 RNA polymerase sequence was purified and cloned into pCAGGS to yield pCAGGS_T7. The sequence encoding the P3 open reading frame (ORF) of FMDV O189 was reverse transcribed using the viral RNA as the template by the action of Superscript III enzyme  with the random hexamer to generate the second helper plasmid. The cDNA was then served as the template to amplify the P3 DNA fragment using primers ClaI_P3_F and NheI_P3_R  at room temperature for 5 min. Simultaneously, 1 µg of pKLS3_GFP and 1–3 µg of pCAGGS_T7 were equilibrated in 250 µL Opti-MEM® I reduced-serum medium.The plasmids pKLS3_GFP, pCAGGS T7, and pCAGGS_P3, were purified using a Mini-Prep purification kit , following the manufacturer’s protocol. In the co-transfection experiment, the pKLS3_GFP and pCAGGS_T7 were transfected directly into BHK-21 cells, while pKLS3_GFP, pCAGGS_P3, and pCAGGS_T7 were combined in the tri-transfection protocol. The combinations of plasmids were incubated with Lipofectamine™ 2000 transfection reagent according to the manufacturer’s instruction  prior to dropping onto overnight seeded BHK-21 cells in 6-well plates. The protocol for co-transfection was optimized by varying the ratio of pKLS3_GFP to pCAGGS_T7 as 1:1, 1:2, and 1:3. In the co-transfection experiment, 10 µL Lipofectamine™ 2000 were incubated in a 250 µL Opti-MEM® I reduced-serum medium. The diluted DNA and transfection reagent were mixed with each other and incubated at room temperature for 20 min. Subsequently, the mixtures were gently added onto the monolayer of BHK-21 cells prior to incubation at 37 °C with 5% CO2 for 5 h. Then, the transfection medium was replaced with Opti-MEM® I reduced-serum medium containing 2% FBS and the cells were incubated at 34 °C with 5% CO2. The mixture of pCAGGS_T7 and pCAGGS_P3 plasmids was also included as a negative transfection control. The co-transfection with pKLS3_GFP and pCAGGS_T7, and a single plasmid transfection with pKLS3_GFP were also performed in parallel to compare the expression level. After 24–48 h post-transfection (hpt), the cells were observed for green fluorescent signals using a fluorescence microscope .In the tri-transfection protocol, 1 µg of pKLS3_GFP, 3 µg of pCAGGS_T7, and 1 µg of pCAGGS_P3 were added to a microfuge tube containing 245 µL Opti-MEM® HD transfection reagent  per the manufacturer’s instructions. Briefly, 1 µg of pKLS3_GFP, 3 µg of pCAGGS_T7, and 1 µg of p CAGGS_P3 were mixed with Opti-MEM® I reduced-serum medium in the final volume of 150 µL. Fifty microliters of FuGENE® HD was then added into the plasmids’ mixture, and the reaction was incubated at room temperature for 30 min to allow the complex formation. The transfection mixture was gently dripped onto the monolayer of BHK-21 cells prior to incubating the cells at 37 °C with 5% CO2 for 4 h. Thereafter, the transfection reactions were removed and replaced with either Opti-MEM® I reduced-serum medium containing 50 µM (EC50) or 160 µM (EC90)  of ribavirin . Then, the cells were incubated at 37 °C with 5% CO2. The fluorescent signals were observed at 24 hpt using a fluorescence microscope . The signal intensity and numbers of green fluorescent cells were compared between drug and non-drug treated units. In addition, rupintrivir, a 3Cpro inhibitor, was tested at concentrations equal to its EC50 (2 µM) and EC90 (4 µM)  to investigate the influence of protease inhibition on the RdRp function.The tri-transfection of pKLS3 and the two helper plasmids was performed on a monolayer of BHK-21 cells in 6-well plates using the FuGENEpol) was studied by means of homology modeling exploiting the platform available on the SWISS-MODEL server  [pol   to eluciwne.pdb, ) was useg Q-MEAN  and Ramag Q-MEAN . The thrg Q-MEAN . The molg Q-MEAN ,37. The g Q-MEAN  with thep < 0.05). The inclusive titers of O189, Asia1, and A/Lopburi were similar. The analysis of the differences between mean titers at each time point is shown in p value less than 0.05. The titer of O189 was approximately 2 × 105 TCID50/mL at 24 hpi; therefore, it was selected for further study.Growth characteristics of three FMDV strains, including type A; A/Lopburi/2012 (A/Lopburi), type O; O/TAI/189/1987 (O189), and type Asia1; Asia1/2013, in BHK-21 cells were determined. Cytopathic effects (CPE) characterized by cell rounding up and increasing reflection were first observed in the monolayers of cells infected with 0.01 MOI of each virus at approximately 12 hpi. The CPE produced by A/Lopburi/2012 was the most noticeable. The complete infected cell lysis appeared at 20 hpi. Viral growth curves were generated by plotting the virus titers against times (hpi). The results showed that the titers of all viruses continuously increased over the 24 h period of infection. FMDV O189 and A/Lopburi exhibited the highest growth characteristics . The oveStuI at the junction between O189 5′ and 3′UTRs. All these cis-acting elements—5′UTR, 3′UTR, and poly A tail—and the hdv ribozyme sequences were located downstream of the T7 RNA polymerase promoter , a stretch of 14 cytosine residues of the poly C tract, and an l-fragment (nucleotides 383-1053) A. The urpromoter A. ReplicThe hdv ribozyme was included in the construct to free the transcribed RNA at its 5′ end. Thus, we tested the function of the ribozyme by an in vitro transcription using the closed-circular plasmid as the template. If it functioned properly, the hdv ribozyme was expected to auto-cleave precisely at its 5′ end. We found that the RNA product generated by the in vitro transcription was approximately 2.2 kb corresponding to the length of the whole O189 cis-acting elements and the hdv ribozyme presenting in the pKLS3 minigenome . The resGFP gene was introduced into pKLS3 at the StuI site as a reporter gene to generate pKLS3_GFP , 3C (protease), and 3D (RdRp). This plasmid DNA was used as a helper vector to supply these proteins in trans. We expected that pCAGGS_P3 would expand the numbers of the transcripts produced by T7 RNA polymerase, serving as the templates for protein synthesis. The results demonstrated that the tri-transfection with pKLS3_GFP, pCAGGS_T7, and pCAGGS_P3 markedly increased both fluorescent signal intensity and numbers of the GFP expressing cells compared to the co-transfection with pKLS3_GFP and pCAGGS_T7 B. AdditiGFP gene. We then examined the application of this minigenome for antiviral drug screening. Ribavirin is a synthetic purine nucleoside (guanosine) analog with a broad-spectrum antiviral activity. It is effective against a large panel of RNA viruses, including picornaviruses, such as poliovirus, FMDV, and enterovirus 71. Inhibition of viral infection occurs through a direct interaction between RdRp and ribavirin-5′-triphosphate. The misincorporation of ribavirin into the viral genome results in increased viral mutation rates, leading to the extinction of the virus population [pro of picornaviruses, such as enterovirus 71 and human rhinovirus [The previous results showed that pCAGGS_T7 and pCAGGS_P3 could function in trans on the pKLS3 minigenome to drive transcription of the pulation . In thisinovirus . BHK-21 inovirus . Thus, tpro inhibitor, which inhibits P3 processing, also decreased the GFP expression. However, the effect of the protease inhibitor was much less pronounced than that of ribavirin. The increased doses of ribavirin and rupintrivir from EC50 to EC90 did not decrease the fluorescent signals much. Please note that the EC50 and EC90 were performed using wild-type FMDV . This finding indicates that our FMDV minigenome system using a combination of pKLS3_GFP and the two helper plasmids is a valuable screening tool for antiviral drugs targeting RdRp and may be 3Cpro of FMDV. Additionally, this platform can be applied to other picornaviruses.The results showed that ribavirin almost completely inhibited the GFP expression as determined by markedly decreased numbers of GFP-positive cells . RupintrThe model of FMDV O189 RdRp was developed, and the quality of the structural modeling was adequate for the molecular docking analysis to confirm whether ribavirin could bind to the active site on RdRp of FMDV. The amino acid sequence identity between the reference and O189 RdRp was 98.94%. The structural quality determined by QMEAN was scored at −0.86, while the Ramachandran plot was favored at 94.23%. The results of randomized docking showed that the ribavirin molecule preferentially buried in the pocket containing the residues Pro169 and Met296 with a binding affinity of −5.4 and −6.4 kcal/mol, respectively . The resViral replicon systems have been developed previously by a number of research groups to study the replication of RNA viruses. Replicons or minigenomes are defined as self-replicating but non-infectious RNAs. Thus, the FMDV minigenome provides an opportunity for molecular biology research related to FMDV replication, transcription, and translation, which has normally been restricted to high biosecurity and containment facilities. Here, we report the development of an FMDV minigenome, pKLS3, which is a DNA-based vector containing the minimum cis-acting elements essential for transcription and translation of FMDV. Because this platform utilizes T7 promoter to facilitate the transcription of the first-strand RNA, the pCAGGS_T7 plasmid expressing the T7 RNA polymerase is a crucial component in this system. Our minigenome possessed a hdv ribozyme downstream from the poly A tail, which auto-cleaved at its 5′end, leading to the transcription termination of the T7 RNA polymerase. Another component, pCAGGS_P3, is not mandatory; however, it acts as the transcription enhancer to increase the yield of the transcripts for protein synthesis by cap-independent translation. The ribosome assembled at the IRES located within the 5′UTR scans through the ribonucleotide sequence and starts reading at the first or second ATG to perform the translation process. Although various FMDV replicons or minigenomes were established, all required in vitro transcription and RNA transfection ,25,26,41For pKLS3 to function as the minigenome, all cis-acting components for the transcription and translation processes, including 5′UTR and 3′UTR, should be intact and work properly. In fact, 5′UTR and 3′UTR interacted together and with other viral and host proteins to form a replication complex. Although the exact component of the replication complex has not been elucidated yet, evidence showed that the cellular poly C binding protein 2 (PCBP2) bound the IRES and interacted with the other host proteins, such as poly-A binding protein (PABP) that attached to the 3′UTR . Translapro. FMDV 3Cpro is responsible for shutting off cellular transcription of host mRNA by cleaving the nuclear histone H3 and inhibiting host protein translation [pro provided in trans mostly interferes with the host protein synthesis by inhibiting cap-dependent translation initiation [pro plays a significant role by cleaving eIF4AI and eIF4GI, the translation initiation factors required by capped mRNA for binding with the 40S ribosomal subunit and unwinding secondary structures of RNA [pro facilitated the translation of uncapped mRNA generated by our pKLS3 minigenome system by hijacking the cleaved eIF4GI for binding with its IRES.Although the helper plasmid containing the FMDV P3 polyprotein, pCAGGS_P3, was not a necessary component for the role of pKLS3 minigenome in the first strand RNA synthesis, it markedly enhanced the GFP expression. In addition to supply the RdRp for the efficient transcription of uncapped RNA produced by T7 RNA polymerase, the P3 also encodes a protease, 3Cnslation . As cellitiation . FMDV 3Cs of RNA . Thus, tThe length of the poly A tail was highly important for the function of the pKLS3 minigenome. We found that the length of poly A tail was significantly related to the level of the GFP expression. One of our constructs containing 19 adenine residues failed to express a detectable level of the green fluorescent signal. Previous studies have shown that 3′UTR and the length of poly A tail of RNA viruses are responsible for viral genome replication ,16. In tXenopus laevis β -globin was generated and used as an mRNA vaccine to immunize mice [Recently, the mRNA vaccine has become one of the most robust technologies to produce vaccines and biomedical therapeutics for emerging infectious diseases and cancers due to its short manufacturing lead time and scalability. The transcribed mRNAs possess the same major advantages as DNA vaccines but lack the adverse effect of DNA integration into the host genome . In addiize mice . Furtherize mice . In theiThus, the development of nucleic acid vaccines and therapeutics are mainly based on DNA molecules. Generally, a plasmid DNA, once delivered into the cells, still requires sequential numbers of intracellular events for DNA translocation from the cell periphery to the nucleus to initiate the transcription. The transcribed mRNA is then transported back to the cytoplasm and consecutively translated into a protein. Since plasmid DNA does not easily pass through the nuclear membranes, only a few DNA molecules can enter the nucleus even in actively dividing cells .We have shown here for the first time the establishment of a DNA-based pKLS3 minigenome system and its application for foreign protein expression and antiviral drug screening. Although pKLS3 and the helper plasmids are DNA, both transcription and translation processes for efficient protein synthesis occur solely in the cytoplasmic compartment, such as the mRNA vaccine. Additionally, handling, storing, and transport of this minigenome system are more convenient than dealing with RNAs. Unlike some DNA and viral vector vaccines, they do not integrate into the host chromosome. With these properties, the pKLS3 minigenome could be one of the attractive candidate vectors for modern biopharma development.pKLS3 vector, its associated products, and the developmental process are protected under the Patent Application Number: 1901006625.