The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology. Deltavirus due to its uniqueness [In 1977, a novel antigen was found in the nucleus of hepatocytes from patients with a more severe form of hepatitis B. It was first thought to be a previously unknown marker of hepatitis B virus (HBV). Only later, it was found that the then called delta antigen was not part of HBV but of a separate defective virus that requires the presence of HBV for infection. The newfound virus was designated hepatitis delta virus (HDV) and, by 1986, its RNA genome was cloned and sequenced  is viral clearance . HoweverHDV superinfection of chronic HBV patients also causes severe acute hepatitis, but in this case, for up to 80% of patients, it progresses to chronicity . The proSince HDV is a satellite virus of HBV, every HBsAgs positive patient should be screened for coinfection with HDV; that is, patients should be tested, at least once, for anti-HDV antibodies. A negative result does not justify testing for HDV RNA as, so far, it seems that every individual infected with HDV develops anti-HDV antibodies . In contCurrently, there is no need for quantification of the HDV RNA levels in serum during the diagnosis step. There is no evidence that a correlation exists between the stage of liver disease and the levels of HDV RNA . Thus, aFor some time, quantification of HDV RNA levels in clinical samples has suffered from the lack of a standardized test. Quantification of HDV RNA was done in specialized laboratories using in-house protocols, which unfortunately become irrelevant outside the laboratory of origin. Such assays typically lacked an internal control and were limited to only one genotype. Furthermore, there is no international reference standard to make results from different laboratories comparable. As proposed elsewhere, an HDV RNA reference preparation should be defined by the World Health Organization to be used as an international standard .In 2012, two standard protocols were proposed to detect and quantify HDV RNA from clinical samples , 33. OneApplication of standardized procedures is crucial to improve our understanding of HDV RNA kinetics during the course of disease. It will improve patient management, as data can be gathered which will help in the decision to start treatment, as well as monitoring the response to therapy in chronic patients. Also it will contribute to the screening of HDV infections in the endemic areas, providing more reliable epidemiological data. Overall, acceptance of standardization will help clarify the pathophysiology of HDV infections.Ideally, a successful treatment of an HDV infection eradicates HDV and its helper virus HBV. Clearance of HDV is obtained when both HDV RNA and HDAg in the liver become persistently undetectable and a complete resolution is achieved when HBsAgs clearance is also obtained.However, at this time, there is no efficient therapy. Prolonged treatment with recombinant interferons is the only therapy that has shown antiviral activity against HDV. Such therapies, which last up to 2 years, have been reported as only 20–40% efficient .In general, when searching for a treatment for viral disorders, the first and preferred targets analyzed are the viral components, such as enzymes involved in the virus replication cycle. But HDV lacks any specific enzymatic function to target. Since the only known enzymatic activity the virus possesses is a ribozyme, the virus relies on the host cell to provide for all other enzymatic activities needed for its life cycle. This represents a serious challenge in finding an HDV-specific therapeutic target.Puzzlingly, the nucleoside and nucleotide analogues used for treatment of HBV infection are inefficient against HDV. Although they block HBV DNA synthesis in chronic patients, they have little impact on HDV and do not even enhance interferon treatments . Famciclα (IFN-α) has been used for treatment of HDV infections since the mid-1980s [α therapy. It seems that 2 years of treatment with IFN-α is superior to shorter treatment durations to obtain HDV RNA clearance [α; patients treated with doses of 9 million units responded better than those treated with only 3 million units, and relapse was common when the IFN-α dose was reduced [α is tolerated by only a minority of patients [α side effects include flu-like symptoms, fatigue, and weight loss as well as severe psychiatric disturbances. Patients have a tendency to become deeply depressed; suicides and attempted suicides have been reported [α dose, and intermittent use of IFN-α, observed in drug abusers, increased incidence and severity of side effects [Interferon-id-1980s . Severalid-1980s . Researclearance . It has learance . The rat reduced . Unfortupatients . IFN-α sreported . The sev effects .α was largely replaced by longer-lasting pegylated IFN-α (PEG-IFN-α) [By 2006, IFN-G-IFN-α) , 41, 42.G-IFN-α) . RibavirG-IFN-α) .α2a alone or with adefovir and adefovir alone [α2a, showing an antiviral efficacy in more than 40% of patients, and 25% became HDV RNA negative [α2a plus adefovir therapy was superior in reducing HBsAgs serum levels [The Hep-Net International hepatitis D intervention trial, which included 90 patients from Germany, Greece, and Turkey, tested PEG-IFN-ir alone . HDV RNAnegative . Adefovim levels .α for one year or longer, if the patient can tolerate the adverse effects of such therapy [Currently, it is usually recommended to treat chronic hepatitis D with PEG-IFN- therapy . For pat therapy .α2a with adefovir to also tackle HBsAgs, crucial for HDV propagation. Most importantly, alternative treatments need to be explored, as the efficacy of the current therapies is clearly unsatisfactory. One of the most promising alternatives is the prenylation inhibitors since, as will be discussed subsequently, prenylation of HDAg is essential for interaction with HBsAgs. Furthermore, prenylation inhibitors have already been developed to treat a number of malignancies and were shown to be safe [An optimization of the available treatment strategies is clearly needed, either regarding doses or duration, and also possible combinations such as PEG-IFN- be safe .An infectious HDV virion is an enveloped, roughly spherical particle, of around 36 nm in diameter . The outSince HDV and HBV share the same envelope proteins, it is often assumed that attachment and cell entry occur via similar mechanisms. Several studies have attempted to identify the regions of the HBsAgs required for HDV and HBV entry. The preS1 region of L-HBsAg is myristoylated at the N-terminus. This posttranslational modification and about 48 adjacent amino acids are essential for HBV and HDV entry into hepatocytes. Synthetic peptides that mimic this region are potent inhibitors of virus entry .Many studies have aimed to discover the host receptors for HBV (and maybe HDV). Many candidates have been proposed but not confirmed .It has been suggested that functional purinergic receptors are required for HDV entry as compounds that block the activation of such receptors inhibited HDV and HBV infection of primary human hepatocytes . Howeverin vitro, using established human liver cell lines, which are much more convenient and reproducible than primary hepatocyte cultures.In contrast to all previous studies, an important new report by Yan and colleagues demonstrates that a necessary and sufficient receptor for HBV and HDV is the sodium taurocholate cotransporting polypeptide . This prHDV has a small circular RNA genome with only ~1700 nucleotides; this sequence length varies by no more than 30 nucleotides among HDV isolates . In natiHDV contains one functional open reading frame (ORF), encoding the delta antigen . This ORThe delta antigen is translated from a third RNA species, a linear 0.8 Kb messenger RNA (mRNA) of antigenomic polarity and a 5′-cap and 3′-polyadenylated tail . The dif6- to 107-fold when compared with uncatalyzed cleavage [3) ribozyme, a conserved mammalian sequence within an intron of the CPEB3 gene [Site-specific self-cleavage and ligation has been reported on antigenomic HDV RNA, showing that this RNA possesses ribozyme activity, just like plant viroids . Both gecleavage , 59. AltEB3 gene . In factEB3 gene .in vitro [HDV RNAs are transcribed in the nucleus of infected cells, but the details of this process remain poorly defined. The three RNA species that accumulate in infected cells are the product of posttranscriptional processing. The precursors, from which they arise, are thought to be transcribed by a double-rolling circle mechanism, exemplified in in vitro . ThroughEven though such a rolling-circle mechanism has been widely accepted as a model for HDV replication, critical details remain to be confirmed and/or clarified such as the host cell components involved (reviewed by ).HDV has no known DNA intermediate, as observed for retroviruses , and theα-amanitin blocks HDV RNA synthesis of both the genomic and antigenomic strands [The host RNA polymerase II (pol II) seems to be required for genomic HDV RNA transcription. Nuclear run-on experiments on an endogenous HDV RNA template have shown that inhibition of pol II by low concentrations of the specific inhibitor  strands . One pos strands . It has  strands . Such elα-amanitin that would inhibit pol II activity [α-amanitin, consistent with pol II involvement [α-amanitin.Despite being shown that pol II interacts with genomic HDV RNA, it has been suggested that a different host polymerase is responsible for the synthesis of antigenomic HDV RNA , 70. Theolvement . Note tholvement . This suin vitro studies, which indicate that fragments of the HDV RNA genome interact not only with pol II but also with pol I and pol III [in vitro interactions may not have biological relevance, especially since they do not lead to RNA-directed transcription.An additional complication arises from  pol III . HoweverThe HDV mRNA possesses characteristics of a pol II transcript that is processed to a mRNA, namely, a 5′-cap structure and a 3′-poly(A) tail. In fact, the role of pol II in HDV mRNA transcription has been generally accepted , 73, 74.The controversy regarding the transcription process is thus limited to whether genomic RNA is transcribed by pol II or another polymerase, either pol I or pol II . If diffIn addition to the posttranscriptional processing to make the abovementioned three HDV RNAs, there is an important RNA-editing event. During the virus replication cycle, some of the antigenomes are edited at a specific site by a host adenosine deaminase (ADAR1). This changes the adenosine in the amber codon to inosine. After subsequent RNA-directed RNA synthesis, it leads to the replacement of inosine with guanosine . That isTherefore, although HDV has only one ORF, it encodes two proteins: the small delta antigen (S-HDAg) of 195 amino acids and the large delta antigen (L-HDAg) with 214 amino acids.The two delta antigen isoforms share 195 amino acids and differ only in that the large form has 19 extra amino acids on the C-terminus. As such, S-HDAg and L-HDAg share several functional domains within the common amino acid sequence, as illustrated in Both delta antigens undergo posttranslational modifications (PTMs) by several host enzymes. Several groups have investigated the impact these PTMs may have on the antigens' functions, but the precise significance of most of these modifications remains uncertain.The exception is one PTM, characteristic only of L-HDAg, which has been shown to be essential. It occurs on cysteine residue 211 and is mediated by a host farnesyltransferase , 83. ThiThere are other PTM events, ones shared by both forms of the delta antigen. These involve phosphorylation, methylation, acetylation, and sumoylation (reviewed in ).Phosphorylation has been observed at multiple sites, mostly at serine and threonine residues. Different phosphorylation patterns were observed for S-HDAg and L-HDAg, and, if relevant, the distinct patterns may in part account for their distinct biological functions . SeveralAcetylation of Lys72 on S-HDAg, by host p300 acetyltransferase, is thought to regulate nucleocytoplasmic shuttling of viral RNA , 92. Notin vitro and has also been proposed to have a switching effect on HDV RNA replication [in vitro. In transfected cells, the mutant S-HDAg reduced genomic RNA synthesis and almost completely suppressed HDV mRNA synthesis [Methylation of Arg13 on S-HDAg, by protein arginine methyltransferase I, has been observed lication , 94. Theynthesis , 94.Finally, SUMOylation of multiple lysine sites, by small ubiquitin-related modifier isoform 1 (SUMO1), has been reported. Such PTM was detected on S-HDAg but not on L-HDAg . And thiAlthough the two delta antigens share sequence and functional domains, they play very distinct roles in the HDV replication cycle. S-HDAg is essential for HDV RNA accumulation, whereas L-HDAg acts as a dominant negative inhibitor of HDV replication  and alsoRegarding the appearance of L-HDAg, it is important to recall that because of the accumulated editing of the HDV antigenome, the proportion of this form, in relation to the total amount of accumulated HDAgs, increases during the replication cycle from 0% to around 30%, . This isS-HDAg has been more thoroughly studied than the large form, likely due to the fact that it is required for the accumulation of HDV RNA. Several roles have been attributed to this viral protein including putative and observed functions.α, since this importin interacts with S-HDAg in vitro [S-HDAg is present in the virions forming viral RNPs with the HDV genome. One of the first tasks it performs is the transport of the viral genome into the nucleus of infected cells, where RNA-directed RNA synthesis takes place. This transport is achieved by the presence of the previously described NLS and RBD. Nuclear import may be facilitated by karyopherin 2in vitro .Another role attributed to S-HDAg is the regulation of HDV RNA editing, particularly the deamination by ADAR-1. This editing seems to occur at multiple locations on HDV RNAs, but it is focused on the antigenomic RNA at the stop codon adenosine . S-HDAg It has been known for more than two decades that the small form of the delta antigen is essential for the accumulation of processed HDV RNAs . Severalin vitro [S-HDAg has been shown to interact with host pol II. In a pull-down assay, both S-HDAg and L-HDAg fused with a glutathione S-transferase tag were able to bind pol II from HeLa nuclear extracts . In the in vitro . Howeverin vitro . They prAmidst all the reports that S-HDAg actively participates in HDV RNA transcription, there is a contradictory result showing that the presence of S-HDAg is not required for the accumulation of processed short HDV transcripts, although full-length transcripts, genomic or antigenomic, do require S-HDAg, or even L-HDAg . As an eIn vitro studies have reported that S-HDAg can stimulate HDV RNA ribozyme activity [in vivo S-HDAg may be directly involved in posttranscriptional processing of nascent multimeric transcripts by enhancing cleavage into unit-length molecules. It should be noted, however, that the abovementioned studies of Lazinski and Taylor indicate that, in vivo, HDAg is not directly needed for ribozyme cleavage and subsequent ligation [Another role attributed to S-HDAg is that of HDV RNA chaperone. activity . From suligation .S-HDAg may also be involved in deviating/redirecting other host cell components to facilitate HDV RNA replication. S-HDAg is a rather promiscuous protein in that many cellular partners have been detected.HDV has a very small RNA genome, as mentioned earlier, and encodes only one viral protein, HDAg. Albeit the fact that a second isoform of the HDAg appears later in the replication cycle, S-HDAg and L-HDAg are not sufficient for HDV to complete its replication cycle. HDV must rely extensively on host cell factors to complete its replication cycle.A comprehensive study using immunopurification followed by mass spectrometry identified over 100 host proteins associated with a tagged S-HDAg . This seHow can one small protein be involved in so many interactions and perform such different functions? The answer may be related to the S-HDAg's lack of structure. Based on S-HDAg's sequence, it has been predicted that the protein is extensively disordered . The preIn fact, S-HDAg has several characteristics generally attributed to IDPs. These proteins are commonly nucleic acid binding proteins displaying chaperone activity, such as S-HDAg. Also, a high net charge is characteristic and S-HDAg has an estimated net charge of +12 . MultimeThe lack of a rigid 3-dimensional structure may account for the protein's promiscuity and ability to take part in several interactions with distinct partners. As such, a deceivingly simple virus, encoding only one protein, can hijack the host cell mechanisms required to complete its life cycle.More than 30 years after its discovery, a lot of fundamental aspects of the HDV life cycle and interaction with the host still remain unknown. But its peculiar simplicity makes all its beauty.


I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke. A power stroke implies movement of a load by reaction to parallel, ATP cleavage-driven motion of a region of the motor. An alternative is movement of a load by thermal motion of the load with ATP cleavage-dependent selection against thermal motion in the “wrong” direction . More generally, biasing of a thermal ratchet can occur via non-specific forces, such as those generated by either electrical fields or pressure gradients. These alternatives have been considered for actin/myosin, kinesin/tubulin, dynein/tubulin, ribosome/peptide and phage DNA packaging motors [i.e., the load) into a cavity of the protein shell of the phage capsid. The shell is typically made of an icosahedrally symmetrical lattice of identical copies of one or, sometimes, more proteins.Analysis of biotic motors  that does have a power stroke –15. SeveAlternative proposals have been made for the delivery of the power stroke. Delivery by the connector is favored by studies of mutants of the connector of phage SPP1 ,17. In ai.e., the sequence of its ancestors, in addition to consideration of the system’s current biochemistry and biophysics [The point has been well made that understanding of any biotic system requires consideration of its history, ophysics ,23. A poophysics –13,18,24Thus, I attempt here to deduce the characteristics of a thermal ratchet-based, DNA packaging ATPase-independent (but connector-dependent and high-energy compound-dependent) ancestor to the type 1, power stroke-dependent motor mentioned above. In so doing, I work with the constraint that the hypothesis be consistent with a reasonable proposal for ancestors of the thermal ratchet-based motor that are ancient enough to be abiotic. Thus, the results include new proposals concerning some general aspects of abiotic  chemistry. The focus on nucleic acid packaging motors has the advantages of (1) the information already obtained for the present-day motors already studied  and (2) the potential information to be obtained, in the future, by detecting and characterizing additional present-day motors that are less evolved than the classical motors .2.2.1.i.e., without known function for cells. Terminase is, therefore, not likely to have been transmitted horizontally. Thus, classical, connector-dependent DNA packaging motors existed before the prokaryote/eukaryote splits, i.e., by about 1.6 billion years ago [Studies of sequence- and structure-based similarity support the assumption that classical phage DNA packaging motors have ∼1.6 billion year-old ancestors that have the components, and presumably the basic mechanism, of the classical motors. These studies include findings of sequence similarity for two of the three critical components of the DNA packaging motors of double-stranded DNA phages and also eukaryotic viruses. These components are (1) the outer shell, for which a variation of one of two basic folds (double β-barrel- and HK97-folds) is found in all studied double-stranded DNA viruses and sequence-based similarity is typically  detected –29, (2) ears ago ,41.A classical, connector-dependent DNA packaging motor begins DNA packaging with the capsid in the procapsid state , after the expansion that occurs during the capsid I to capsid II transition, i.e., after the procapsid to mature capsid transition [in vivo [in vitro phage ϕ29 DNA packaging [in vitro packaging [The need for an explanation for shell flexibility has recently increased with our finding that a second shell expansion occurs during phage T3 DNA packaging in an infected cell (ansition . This se[in vivo . Our int[in vivo ). Re-staackaging  and by iackaging ,48.2.2.Cystoviridae. The Cystoviridae include ϕ6, ϕ8, ϕ12 and ϕ13 . Thereviews: ,51.Tectiviridae, another variation of packaging-associated expansion occurs. In this case, the protein shell of the capsid surrounds an inner shell of phage protein-enriched host membrane. This membrane forms the cavity in which DNA is packaged. Packaging is through a unique outer shell vertex and a packaging ATPase has been found. However, a connector has not been found [In the case of the double-stranded DNA-containing en found . Althougen found . Thus, aCystoviridae, has no known selective advantage. Additional room could have evolved more simply with an original shell that had a larger radius. In fact, most hypotheses for packaging ATPase-based motors do not even include the shell expansion as part of the motor [When one considers only the biochemistry and biophysics, packaging-associated expansion of the nucleic acid-enclosing region of the viral capsid, which occurs for almost all known double-stranded DNA phages and the RNA-containing he motor –15. ThusThus, in the present exercise, I use the shell expansion as a point of departure for a hypothesis that describes the nature of abiotic ancestors to modern viruses (and cells). The hypothesis includes a proposal for abiotic ancestors that occurred before the advent of polymer coding, which was perhaps 3.8–4.5 billion years ago . Among t3.In the present communication, the objective is to deduce a plausible sequence of ancestors, beginning with abiotic ancestors and ending with a classical DNA packaging motor. Importantly, at least some aspects of a proposed ancestor sequence are expected to be testable via the isolation of phages from niches that favor pre-classical nucleic acid packaging motors. The theory has the potential to provide clues to where and how to find living versions of such anciently derived phages. Obviously, assumptions will have to be made because neither the abiotic ancestors nor the early biotic ancestors have been observed.Thus, the above data are complemented here by the following plausible assumptions. First, I make the relatively generic assumption that the earliest ancestors are completely abiotic in character. That is to say, the earliest ancestors occur for reasons of chemistry/geology only and do not include a component of information-carrying polymers. This assumption excludes consideration of the arrival of information-carrying polymers from an extraterrestrial source, another planet, for example (exogenesis). If, nonetheless, exogenesis did occur, the abiotic ancestors proposed here are considered to occur at a location other than planet Earth. The onset of biotic ancestors for DNA packaging motors presumably intersects the onset of all biotic ancestors.Second, I make the assumption that the earliest ancestors occur in regions of the Earth that have a relatively high concentration of molecules in general and of precursors for “biotic” molecules in particular. The more concentrated the precursors, the higher the probability that productive, but random and rare, events occur in succession, as required for the onset of biotic events. As discussed elsewhere, this region of the Earth is likely not to be at the surface  and Third, I make the assumption that all energy transduction of the earliest ancestors occurs via changes in chemical bonding that occur relatively frequently; changes in non-covalent bonding may be involved. This assumption is based on the notion that the most frequent changes in either covalent or non-covalent bonding are the most likely to generate, by chance, the non-information-dependent (abiotic), earliest ancestors.i.e., by geological forces and diffusion. The rationale for this assumption is that transport increases the probability of productive, although uncontrolled, abiotic changes by increasing the diversity of the compounds with which any given compound comes in contact. The protective aspect maximizes the potential for preserving relatively unstable molecules during transport. This assumption introduces the concept of transport-dependent chemical potential gradients, a concept that differs from the current consensus of chemical potential gradients that do not depend on transport .i.e., the RNA cells discussed below. That is to say, even though not yet motile, the ancient biotic systems moved enough for content exchange sufficient to bring all of them in contact with each other. The explanation for the near-universality of the genetic code concludes with the highly plausible assumption that this exchange was essential to convergence of initially arising codes to an optimized code that is empirically known to be  universal [The fourth assumption is indirectly justified by its explanation of two otherwise paradoxical observations. These two observations are near-universality of the genetic code and unique chirality of biotic molecules, for example, sugars, amino acids and nucleotides ,58. The niversal ,56. WhenFifth, I re-state the assumption that the classical nucleic acid packaging motors studied thus far retain enough characteristics of their abiotic ancestors so that one can use the classical motors as a starting point for deducing the characteristics of the abiotic ancestors. This fifth assumption is the most uncertain assumption, of course. It is rooted in the conclusions (discussed above) that (1) the components of today’s DNA packaging motors have ancient origin, (2) capsid expansion associated with nucleic acid packaging is an unexplained phenomenon that is potentially rooted in the ancient past, and (3) a thermal ratchet-based motor is likely to have been one of the earliest ancestors. In other words, the fifth assumption is that some aspects of classical nucleic acid packaging motors are imprints  from an ancient period . 4.4.1.The hypothesis starts by proposing that, for both viruses and cells, the earliest abiotic ancestors are hollow, porous shells that are unanchored and, therefore, capable of being transported. These abiotic shells (1) are made of geologically generated compounds that happen, because of their chemistry and surrounding conditions, to have an internal cavity, (2) exist in a region of highly concentrated organic molecules, some of which are ancestors of biotic polymers, and (3) have open regions (pores) so that the internal cavity can be filled with one or more of the organic molecules. Although shells of this type appear not to have been observed, related shells have been observed for rare earth borides. The rare earth borides form crystals of interconnected octahedral and icosahedral shells of boron, with details dependent on the molar metal/boron ratio. For some of these compounds, a single rare earth metal atom is packaged at the center of a shell ,61. The The proposed abiotic shells of The hypothesis incorporates assumption 5 by continuing with the following. Among the various abiotic, transporting shells, some are, by chance, part of an abiotic, chemical energy-driven system that makes the shells more transport active via closing of the pores. The chemical energy comes from the binding of relatively high-energy compounds  displacement by the high-energy compound of molecules bound at a pore  omnipresent and keeps the shells sealed while the packaged molecules are transported among regions of relatively low packaged molecule concentration .The enhanced transport eventually brings the shells to a region in which the high-energy compound has a relatively low concentration. When that happens, the high-energy compound dissociates from the shells, thereby causing contraction and permeability increase of the shell. The packaged molecules now diffuse into a new environment. Thus, the range of potential reactions is increased in relation to the range that existed without transport. Although inefficient in promoting chemical diversity by the standards of biotic events, the proposal here is that these energy-transducing, transporting shells provide the most efficient abiotic means for promoting synthesis of ancestor compounds in a sequence of events that ends with production of information-carrying polymers.i.e., no polymer coding-dependent) event exists at this point. Methane is the likely high-energy compound, given that methane is potentially generated throughout most of the geosphere via abiotically produced hydrogen  and the Fischer-Tropsch reaction (review: [I emphasize that the ancestors in (review: ). Sugges can occur only through an abiotic process that resembles a biotic process. To progress from the scenario of The next stage in the succession of ancestors is like the previous stage , except After the shells return to the condition of i.e., a compound represented by red spheres in I propose, however, that a biotic stage is approaching via dimerization of the reaction product compounds. A relatively featureless monomer of a reaction product compound . Finalli.e., cells with components dependent on both RNA catalysis and RNA-encoded information) in the next section.As time progresses, the packaged, quasi-palindromic molecules become increasingly more active in catalysis. Again, the basis for this change is change of the palindromic nature of these molecules, which gives them the secondary and tertiary structure needed. Progression accelerates again when the packaged ancestor molecules have catalytic activity that is expressed while packaged. Nonetheless, the ancestor progression is still abiotic in character, although conditions now exist to produce a molecule that not only has enzymatic activity, but also enzymatically duplicates by initially serving as a template for producing a complement to itself. Again, without proposing further details, I make the leap to RNA cells  no cellular biotic membrane evolved for optimization of uptake and release of compounds, (2) enzymatic activity provided only by ribozymes. and (3) information limited by the relative instability and, therefore, short length of RNA molecules. To give the RNA cell a more concrete basis, the proposal has previously been made that, initially, the envelope of RNA cells is abiotic in character. Possibilities include FeS walls that currently exist in hydrothermally formed iron sulfide chimneys under the oceans ,56,57,76Here, I propose adjustment of this idea by the following continuation of the proposed sequence of ancestors, above. The envelope of the simplest RNA cells is a composite of RNA and abiotic shell. This composite is schematically  illustrated in i.e., via evolution) so that those RNA molecules that provide a selective advantage are selectively retained when pores open. The metabolism, including templated RNA replication, occurs in the cavity of the shell. The retention of vital components during exchange with the environment is a characteristic that forms part of the foundation for further evolution.In addition, RNA cells become more complex when aggregation and, then, merger occur among the simpler RNA cells. Merger causes mixing of packaged RNAs and other molecules, via opening of pores. Merger also causes mixing of RNAs that are integrated in the shell. I note that these RNA cells are still obtaining RNA precursors by the relatively inefficient process of Although polymer-encoded information and polymer-catalysis are both already part of what is now a biotic system, I note that capacity for transmission of the RNA-encoded information is limited by the absence of a shell that replicates along with the RNA and assists the intake of RNA precursors. That is to say, RNA replication occurs within a shell with no classical method for vertical transmission. Instead, vertical transmission occurs via recruitment of new abiotic shells by RNA molecules that have been synthesized in other shells and have been either extruded into the environment or transmitted via contact. In some cases, this recruitment involves displacing RNA molecules previously present. Thus, along with catalysis and polymer-encoded information, competition is also part of the RNA world at this stage.In anticipation of the eventual production of protein-synthesizing cells, one imagines (in an embodiment simplified for illustration) that a RNA cell with a packaged, incipient precursor to tRNA merges with two other RNA cells, one with an incipient precursor to rRNA and the other to mRNA. This merger is an accident and is not guided by complementarity of the shell-incorporated RNA molecules. The result of the merger is an RNA cell that is a potential ancestor of a cell that has protein synthesizing capacity. As hazy as the details are, this idea does produce the concrete prediction that the first mRNA has palindromic character. Other mergers produce precursors of some viruses, although other viruses have single-shell ancestors.Viruses and cells diverge from each other in the RNA world. A virus is differentiated from a cell in that the RNA incorporated in a virus shell has complementarity to some, but not all, RNA of a more complex RNA cell. That is to say, the virus has RNA molecules associated with at least one, but not all, of the shells that merged to form the RNA cell. Thus, the virus recognizes the RNA cell via complementarity of shell-incorporated RNAs.The result is templated merger of the shells and their contents with outcome varying between two extremes. The first extreme is that the virus RNA out competes the other RNAs of the RNA cell and basically converts the RNA cell to a virus. This corresponds roughly to the classical infection with a lytic phage. The second extreme is that the virus provides a function that assists the RNA cell in becoming more efficient and competitive in propagating itself, still by the non-classical process described above. This outcome corresponds roughly to the classical infection with a temperate phage, but with more impact on the cell than is usually attributed to classical infection with a temperate phage. An infection of this type is basically a merger of the type that created RNA cells.4.5.The hypothesis continues by proposing that, as a result of the various shell mergers and the reactions promoted by them, RNA cells acquire protein-synthesizing capacity and a biotic envelope. After first acquiring protein-synthesizing capacity, cells progressively substitute protein and other macromolecules for abiotic and RNA components of the shell until a protein-containing, completely biotic shell (cell membrane) evolves. Details are not proposed here. Viruses co-evolve a biotic shell because viruses are selected for efficiency of infecting whatever cells exist. Some details are discussed in the next section. Before the cell membrane replaces the abiotic component of the shell of RNA cells, exchange with the environment occurs by the process of Cystoviridae) does provide some support for this scenario. Specifically, the genomic segmentation of the Cystoviridae is accompanied by capsid segmentation to the extent that the various compartments of the capsid expand independently as their genome is packaged (discussed above). The capsid segmentation suggests an ancient merger of shells to form a single virus particle. The single virus particle retains the advantages of merger by converting merged shells to a single partitioned capsid and packaging multiple RNA molecules.Empirically, the existence of some RNA viruses that are segmented  viruses still exists today and retains the imprint of compulsory aggregation. In fact, when we used techniques appropriate for isolation/propagation of aggregating phages [In addition, merger via either RNA-directed or random aggregation is likely to be endemic for both cells and viruses in the RNA world, for the following reason. Information in the RNA world is almost certainly not either transmitted or used with precision ,57,72. Tg phages , we foung phages . Genomicg phages .Cystoviridae. As discussed above, Cystoviridae convert each of several packaged single-stranded RNAs to double-stranded RNAs while the RNAs are in the packaged state. This conversion might be considered surprising, if one does not consider the ancestry of viruses. However, the proposal made here is that this ancestry includes RNA world viruses that had (limited) capacity for ribozyme-dependent metabolism. Therefore, the retention of enzymatic capacity is understood as an imprint that survived the replacement of ribozymes with protein enzymes.Another apparent imprint from RNA world viruses is the following characteristic of Parenthetically, I note that, even if the total number of phage particles in aggregates is high enough to detect by almost any procedure, the physical properties of phage aggregates make them difficult-to-impossible to detect by conventional procedures. The reason is that the aggregates are either lost during the removal of cells from a preparation or have properties so different from those of conventional single virus particles that they are not distinguished as virus-like. Biofilm-like phage aggregates have been propagated and detected via needle transfer of particles in zones of bacterial clearing in dilute (<0.1%) agarose gels. The constituent phages sometimes aggregate to the extent that they do not form single phage-originated plaques ,78.4.6.The hypothesis continues by proposing that the following occur as RNA cells acquire the capacity for synthesizing peptides via an RNA template: (1) substitution of peptides for RNA in the shells of i.e., what we now know as the shell of a viral capsid and (2) a cellular membrane that forms the envelope of the first completely biotic cells. The ancestor branch leading to completely biotic cells does not retain the original abiotic shell symmetry, but the ancestor branch leading to modern viruses does.The hypothesis again continues by proposing that formation of these peptide dimers begins a process that continues to more extensive multimerization of peptides. In the case of both cells and viruses, peptide multimerization eventually produces a composite shell with protein molecules, and possibly some residual RNA molecules, in an array that physically is missing the abiotic shell component, but informationally is imprinted with the shape and symmetry of the abiotic shell. Subsequent divergent evolution produces (1) an all-protein viral shell, At least in part, the reason for this difference is that the most immediate RNA world ancestors of completely biotic cells are really each a product of the fusion of several shells. In contrast, the most immediate RNA world ancestors of completely biotic viruses are either the products of no fusions or the product of relatively few fusions, thereby reducing divergence from the symmetry of the original abiotic shells. In addition, virus infection becomes less contributory and more parasitic as cells evolve more capacity for synthesis and metabolism and are less dependent on outside input. Thus, viruses are not as selected as cells for divergence from the original symmetry. Nonetheless, even today, viruses still contribute at the genomic level and cannot be considered purely parasitic –84.Papillomaviridae, SV40 and Polyomaviridae, have an icosahedral arrangement of pentamers. The all-pentamer aspect implies that local symmetry does not exist even though global symmetry does [i.e., a reflection of history, not a reflection of the biophysically most efficient mechanism of assembly given present circumstances.The ancestor succession proposed in the previous paragraph, although obviously missing many details, does have some empirical support. First, this succession predicts that the original scaffolding for viral shells is an abiotic shell that is at least sometimes symmetrical, based, in general, on the tendency for inorganic ions to form symmetrical structures  and, in try does –87. Thatarchaeal viruses do not have either icosahedral or comparable symmetry and include spindle-shaped, bottle-shaped and droplet-shaped capsids [In support, some capsids of double-stranded DNA  capsids –90. The  capsids , separat capsids –94. The I note, however, that the proposed ancient divergence of cells and viruses does not preclude the subsequent reductive evolution, assisted by horizontal gene transfer, of some cells to viruses. Such reductive evolution could begin, for example, by parasitism of one cell by another and could continue by the progressive loss of unnecessary functions by the parasite . This coIn conclusion, the concept of a common ancestor to viruses and cells has itself an ancestor in the proposal that viruses are the building blocks of cells. This latter proposal was made before the onset of modern molecular biology . Spe4.7.The introduction of DNA genomes is essential to increasing the coding capacity of cells, because of the instability of RNA, as discussed above. The hypothesis has been previously been presented that the transition to DNA genomes begins as a viral defense against host ribozymes that degrade RNA viral genomes. The viral use of a DNA genome is, by this hypothesis, then transferred to cells ,99. The Given the potential for increased information storage within a DNA (rather than RNA) genome, realization of that potential evolves for viruses, in part, by increasing the density of DNA packaged in the cavity of a viral shell. This evolution occurs by selection for the following two changes: (1) elimination or reduction of the amount of RNA and other non-DNA molecules in the cavity of the viral shell and (2) developing of both a mechanism and an energy source for condensing a DNA molecule to a higher density. The first change requires, in turn, a gatekeeper that selectively allows entry of DNA and restricts entry of other molecules.Thus, the hypothesis continues by proposing that selection for increased information carrying capacity produces the connector by selection for (and evolution of) a gatekeeper. This occurs before evolution of the DNA packaging ATPase. The connector recognizes and binds the end of a double-stranded DNA molecule . Recogniin vivo to form a procapsid shell in the absence of the connector. This observation was originally made for phages T7 [in vivo studies of [in vitro studies. The reason is that nucleation of procapsid assembly is likely to be at least a fifth order reaction, which requires the conditions of in vitro studies  to be much closer to in vivo conditions than is likely to be achieved for an in vitro system without impractically large effort.The following observation is support (not proof) for evolution of the connector protein after the shell protein, even though sequence similarity is more readily observed for the shell protein –29 than hages T7 ,106 and hages T7 . Nonetheudies of –107 are In The process of The DNA molecule will be injected into cells by a relatively inefficient mechanism related to the mechanism of expulsion of packaged molecules in After appearance of the motor of 4.8.These different viruses include the single-stranded RNA viruses that assemble without a protein scaffold and with assistance from the RNA genome in regulating subunit conformation. Included are several plant and insect viruses, as well as phages such as MS-2 and R17 (reviews: –110). AlThus, a spin-off of the basic hypothesis is that RNA/protein co-evolution produces an alternative mode of assembly for some single-stranded RNA viruses. In this alternative mode, single-stranded RNA forms a hydrogen bond-rich tertiary structure that serves as either a scaffold or a chaperonin for the subsequent assembly of protein subunits to form shells . That is to say, the RNA-scaffolding viruses have capsids with shells that are more diverged from the original abiotic shells than the shells of viruses that use protein scaffolds.Phages with single-stranded DNA genomes have yet another packaging system. These phages have relatively few representatives among isolated viruses , the firOther single-stranded DNA phages have a packaged DNA molecule folded on itself inside of a filamentous protein capsid . I 4.9.To achieve the high DNA packing density of classical double-stranded DNA phages , ev(1) As proposed for the ancestor virus of (2) At this point, the more evolved virus does go further because further evolution has produced signaling capacity that is activated by slowing/stalling of packaging and causes two co-evolving changes in the response to stalling at the point of In the transition illustrated by i.e., the shell conformation of After the contraction, the shell spontaneously returns to its ground state, The cycle of i.e., clamping of the DNA molecule) is the simplest type of connector closing, binding of the DNA molecule works as well. In support of an anti-DNA expulsion role of the connector during packaging, an anti-DNA expulsion role of the connector is known to exist in classical phages after DNA packaging is completed .Finally, I emphasize that viruses with this pre-classical motor do not use a DNA packaging ATPase. I repeat that the basis for proposing the cycle of 4.10.The hypothesis concludes by proposing that the DNA packaging ATPase initially evolves to make the thermal ratchet-based motor of The adoption of a second, specialized protein is selected because of the following two advantages: reduction of the effects of thermal noise on the signaling of the motor of in vitro systems). However, this further evolution does not eliminate the type 2 cycle because selective pressure for the packaging-associated removal of non-DNA molecules remains. Thus, the hypothesis concludes by proposing that the type 2 cycle of classical DNA packaging motors is activated whenever the type 1 cycle both stalls and does not re-start by itself, as discussed above.The final result is the evolution of the ATPase/connector-, power stroke-dependent, classical type 1 cycle discussed above. The type 1 cycle displaces the type 2 cycle at the beginning of packaging and possibly continues without intercession of the type 2 cycle when packaging occurs without the high concentration of non-DNA molecules that are in a bacterial cell  An observed hyper-expansion of the shells of T3 DNA packaging intermediates  is an im5.5.1.Although not an essential aspect of the hypothesis presented here, the location of abiotic ancestors is a peripheral aspect for which some information does exist. Several lines of reasoning have led to the proposal that abiotic ancestors of current biotic systems are located either below the surface of the solid portions of the earth or at the ocean floor. The formations generated by hydrothermal vents are one specific possibility. The lines of reasoning include the difficulty in imagining anywhere else where both high concentrations of abiotic precursors and adequate chemical potential gradients exist The first test proposed here is a search for either the proposed abiotic shells or their fossils. The major problems of such tests will be (1) preserving shells, especially if they were formed and exist at high pressure, (2) establishing an ancestor succession, even if the shells are found and have interesting contents. The latter might be possible by radiological dating of the shells, if found, and their contents. Tests of this type have the disadvantage that a negative result is not conclusive because of the possibility that the shells either are lost during the test or no longer exist.The second test is a search for pre-classical, biotic ancestors. Our recent finding of aggregating RNA viruses ,78 raisei.e., phages whose characteristics are less diverged from ancestral phages than those of the classical double-stranded DNA phages. This venture may be possible with current technology for the following reason. The less diverged phages will have a DNA packing density lower than that of the classical phages, which, thus far, are almost identical in DNA packaging density  [The third test targets the accuracy of the most uncertain assumption made here. This assumption is that an observable phenomenon, the phage DNA packaging motor, retains some characteristics of the earliest ancestors of all living systems (assumption 5). A test of the accuracy of this assumption also appears to be the isolation and characterization of ancestors, in this case living versions of pre-classical double-stranded DNA phages,  by DNA) ,116.Because the hydration of unpackaged DNA is relatively large (∼3 g water/gm DNA) in media with relatively low water activity, the hydration of packaged phage DNA increases as the DNA packing density decreases in these media . Based o6.The following is a summary of the components of the overall hypothesis presented here.6.1.The synthesis of precursors of biotic polymers begins with transport-dependent development of chemical potential gradients. The transport occurs in abiotic shells that both package and protect the precursors. These shells move via geologically derived forces.6.2.Some shells have an expansion/contraction cycle that makes molecule packaging and, therefore, transport-driven synthesis more efficient. The expansion/contraction cycle is driven by binding/releasing of a high-energy compound, possibly methane hydrate. Binding causes the expansion, possibly via cooperatively increased hydration of the shell-forming atoms. An imprint of the characteristics of these shells is transmitted to the viral capsids of today.6.3.The transport-driven synthesis acquires autocatalytic character when the products of this synthesis stimulate post-transport release of their precursors from shells. This process advances when products become part of the shell and dimerization of the products stimulates release of the precursors.6.4.The dimerizing products acquire palindromic character, which is the foundation for the acquiring of both enzymatic activity and complementary pairing of single chains. The introduction of palindromic character is potentially the missing link that resolves a previously presented paradox of feasibility  for the 6.5.The reactions of transported molecules occur in the interior of the transporting shells, which now transport not only the original precursors, but also products. These reactions become enzymatic in character, as the palindromic character of some of the products introduces the complexity of structure needed for enzymatic activity. The first templated replication occurs.6.6.After a gap in the sequence of proposed ancestors, abiotic shells with associated RNA become RNA cells and viruses. Aggregation-derived redundancy is the initial foundation for bypassing limitations caused by the inaccuracy of RNA-based information transfer. Post-aggregation fusion generates a protein synthesizing system and additional complex biochemistry. Fusion is also the source of the progressive differentiation of viruses from cells. A virus for any given cell is a shell (with shell-integrated RNA) that (a) has a copy of one of the shell-associated RNAs that previously was part of a shell that fused to form the cell to be infected, and (b) therefore recognizes the cell via complementary base pairing of RNAs. Generation of complexity via fusion is a version of horizontal transfer, previously proposed to be a means for early evolution . In the 6.7.The hypothesis leaves another gap at the formation of the first biotic cell membranes and cell walls. Possible chemistries involved have been previously discussed .6.8.After emergence of DNA genomes, double-stranded DNA viruses initially package a genome via shell expansion that is an imprint from the abiotic shells. Single-stranded nucleic acid viruses evolve away from this imprint because of the capacity for nucleic acid/protein co-evolution introduced by palindrome-based structures of single-stranded nucleic acids. The shell expansion-driven packaging of double-stranded DNA evolves to acquire thermal ratchet character, via selection for increase in both the speed of DNA packaging and the amount of DNA packaged. The thermal ratchet includes a connector that provides a DNA bind/release cycle. The bind/release cycle is interleaved with a shell expansion/contraction cycle that results in the packaging of a DNA genome, coupled with the expulsion of non-DNA molecules that had been accidentally packaged.6.9.The thermal ratchet evolves a DNA packaging ATPase, via selection for the increased efficiency of dividing functions between two proteins. The DNA packaging ATPase subsequently evolves a power stroke via selection for the increased speed and force of a power stroke-based (type 1) cycle. But, the thermal ratchet-based cycle is retained as a “back-up” (type 2) cycle because of selection for its capacity to increase the density of packaged DNA by expelling accidentally packaged non-DNA molecules.


Sequence determines the three-dimensional structure of RNAs, and thereby plays an important role in carrying out various biological functions. RNA duplexes containing Watson-Crick (WC) basepairs, interspersed with non-Watson-Crick basepairs, are the dominant structural unit and form the scaffold for the 3-dimensional structure of RNA. It is therefore crucial to understand the geometric variation in the dinucleotide steps that form the helices. We have carried out a detailed analysis of the dinucleotide steps formed by AU and GC Watson-Crick basepairs in RNA structures (both free and protein bound) and compared the results to that seen in DNA. Further, the effect of protein binding on these steps was examined by comparing steps in free RNA structures with protein bound RNA structures.Characteristic sequence dependent geometries are observed for the RR, RY and YR type of dinucleotide steps in RNA. Their geometric parameters show correlated variations that are different from those observed in B-DNA helices. Subtle, but statistically significant differences are seen in roll, slide and average propeller-twist values, between the dinucleotide steps of free RNA and protein bound RNA structures. Many non-canonical cross-strand and intra-strand hydrogen bonds were identified that can stabilise the RNA dinucleotide steps, among which YR steps show presence of many new unreported interactions.Our work provides for the first time a detailed analysis of the conformational preferences exhibited by Watson-Crick basepair containing steps in RNA double helices. Overall, the WC dinucleotide steps show considerable conformational variability. Furthermore, we have identified hydrogen bond interactions in several of the dinucleotide steps that could play a role in determining the preferred geometry, in addition to the intra-basepair hydrogen bonds and stacking interactions. Protein binding affects the conformation of the steps that are in direct contact, as well as allosterically affect the steps that are not in direct physical contact. The double helical structure of nucleic acids exists in various polymorphic sub-states, of which DNA prefers B-form conformation and RNA prefers A-form. The A-form in RNA is a right-handed helix formed by stacking of Watson-Crick (WC) basepairs along with a few non-Watson-Crick (NWC) basepairs. The overall conformation of the helices is dictated by the geometry of successive dinucleotide steps, which in-turn is dictated by the chemical nature of bases involved in forming these step. A-form helix is characterized by large roll angle and negative slide values, compared to B-form and is accompanied by a narrow but deep major groove and a wide, shallow minor groove. Significis orientation (cisWW family), as well as basepairs involving the Hoogsteen and Sugar edges. In addition, other non-canonical basepairs from the cisWW family e.g. GU, GA, UU also occur frequently in the RNA duplex[The helical regions in DNA are comprised almost exclusively of canonical Watson-Crick (AT and GC) basepairs, which form 10 unique dinucleotide steps. On the contrary, an RNA duplex consists of canonical WC basepairs (AU and GC), that are paired along Watson-Crick edge in A duplex-11. ThusThe diverse RNA structural motifs and equally diverse proteins that interact with RNA suggest that the conformational changes that occur during RNA-protein interaction can be characterized by changes in the protein, the RNA, or both. An analab initio modeling of RNA structures.The geometry of dinucleotide step is mainly influenced by basepair hydrogen bonds and stacking interactions-24. AparIn this work, a non-redundant RNA crystal structure dataset has been created and we have examined the intrinsic geometries of all WC basepairs and the dinucleotide steps formed by them in the helical regions. To understand the extent of conformational variability that can occur in the dinucleotide steps, the effect of protein binding on these steps was examined by comparing the helices in free RNA with protein bound RNA dataset. Further, we have carried out a systematic analysis of dinucleotide steps to identify potential hydrogen bond interactions between all four bases and correlated the occurrence of such bonds with the dinucleotide step geometry.‘free-RNA’ dataset and those that were in complex with a protein were grouped as ‘bound-RNA’ dataset  was generated using NUCGEN[ModelRNA’) in our analysis for a more realistic comparison with crystal RNA datasets.The x-ray crystal structure dataset was created by extracting structures with resolution better than 3.0 Å from the Protein Data Bank. The datt Table . A non-rg NUCGEN. Unlike g NUCGEN,42. We hThe helical structures in each of the datasets were subjected to geometry based identification and classification of basepairs using BPFind program with default criteria. HelicalHydrogen atoms coordinates was added to all the crystal structures using REDUCE program. The folbound-RNA dataset, the interactions between the atoms in a dinucleotide steps and the protein atoms were identified using CONTACT program in CCP4 program suite[cont) and those that are not in contact (non-cont). In order to assess if the difference in step parameter values between the various datasets was significant, an unpaired student-t-test was carried out.In the am suite. A contaMATLAB was used for all statistical analysis and for plotting graphs.free-RNA dataset consists of 88 protein-free x-ray crystal structures, while bound-RNA dataset includes 127 structures / Pyrimidine-Pyrimidine (YY), Purine-Pyrimidine (RY) and Pyrimidine-Purine (YR). The mean and standard deviation values of the step parameters, along with the corresponding average propeller-twist, cup and Zp values, for the three types of dinucleotide steps in the free-RNA and bound-RNA dataset are tabulated in Tables ModelRNA are also listed. A comparison of the crystal structure geometries with the model structure values indicates that these steps show some characteristic sequence dependent preferences in their geometries.The intra-basepair parameters of WC basepairs present in the RNA datasets are comparable to that of the model structure but are characterized by large variations. The only noticeable features are that GC basepairs have higher negative buckle compared to AU basepairs, while AU basepairs show higher open angle value compared to GC Table . OverallModelRNA value  and lower for steps with only GC basepairs . Roll value differs between RR, RY and YR steps (YR > RR > RY), though the overall average roll value for WC basepair containing steps is lower than e Table . It is iADNA and BDNA did not show any such difference when compared to other RY steps. All three YR steps have larger roll and negative slide values when compared to the RR and RY steps, as well as the ModelRNA  with those of ADNA and BDNA datasets in order to identify correlations that are specific to A-form helices. The parameters that show statistically significant correlations at confidence level > 99.9% being discussed further  have smaller negative values compared to free-RNA .The mean and standard deviation values of intra-basepair parameters for canonical AU and GC basepairs in the t Table . Almost bound-RNA dataset are in direct contact with protein (Table cont) and those that do not contact the protein (non-cont). The mean and standard deviation values of the step parameters and the corresponding average propeller-twist, cup and Zp value, for each of the 10 dinucleotide steps in the non-cont and cont dataset are tabulated separately in Tables non-cont and cont dataset are quite similar to each other, but differ slightly from free-RNA values. Interestingly both cont and non-cont data show large standard deviation values. In addition, the correlation analysis for dinucleotide steps in the bound-RNA dataset does not show any significant difference from that of free-RNA and ADNA datasets  unpaired student t-test was carried out  from the free-RNA dataset. Some of the steps also showed significant difference between free-RNA and non-cont dataset.To check for statistical significance of the differences between the step parameters of the three datasets  while, unlike in BDNA, there is very little overlap in strand II (Pyr-Pyr stacking) and no overlap between the cross-strand bases. RY steps show high intra-strand base overlap along both strands I and II and no cross-strand overlap, with the exo-cyclic atoms making substantial contribution. YR steps in RNA and ADNA datasets are characterized by very small intra-strand contribution and stacking arises mainly due to cross-strand overlap of purine bases, with contributions from both ring and exo-cyclic atoms. Interestingly the overlap in free-RNA crystal structure steps is smaller than in the ModelRNA, for RR and RY steps. Our findings suggest that the combined effect of large negative slide and lower twist value contribute towards these overlap patterns, indicating that the interactions that determine the base stacking preferences of dinucleotide steps in RNA helices are different from those seen in DNA helices. We have therefore analyzed various dinucleotide steps to see whether the base overlap patterns are related to formation of some potential non-canonical, intra-strand or cross-strand hydrogen bonds.Since the parameters for RR, RY and YR steps show some significant differences from s Table . Figure free-RNA dataset is given in Table ModelRNA as compared to fibre-BDNA model. Many of these are retained, with improved hydrogen bond parameters, in the RNA crystal structures, while some potential interactions are found to occur in specific dinucleotide steps.Some of the geometric preferences seen in dinucleotide steps of DNA have been attributed to the presence of additional hydrogen bond interactions between the bases, particularly in oligo-A tracts,34,35. SfreeRNA, on the minor groove side , as well as AA/TT and GA/TC steps in BDNA. The cross-strand N-H..O interaction between 6-amino group of Adenine and O4 atom of Uracil that is commonly seen in AA/TT steps of BDNA (89%) is favoured only in 40% of the AA/UU steps in freeRNA dataset. In GG/CC steps, intra-strand N-H..N interaction between the two Cytosine 4-amino groups shows significant presence in all A-form structures. It is not present in fibre-BDNA model but is seen in BDNA crystal structures with a slightly longer donor-acceptor (DA) distance, as compared to RNA structures. Though a similar pair of 6-amino groups of Adenine is present in AA/UU, they do not have favourable hydrogen bond geometry.Among RR steps, cross-strand C-H..O hydrogen bonds are found in 85% and 59% of AA/UU and GA/UC steps respectively in  Figure . SimilarModelRNA and fibre-BDNA model does not have a favourable geometry for this hydrogen bond. In addition, cross-strand interactions between the two purine bases are highly favoured in AC/GU and AU/AU steps in all RNA datasets and equivalent steps in DNA. A cross-strand N-H..O hydrogen bond is present in 83% of AC/GU in free-RNA, between 6-amino group of Adenine and O6 atom of Guanine. An even larger number (~90%) of AU/AU steps show cross-strand N-H..N interaction between the 6-amino group of Adenines, in both free-RNA and BDNA datasets. A combination of high negative average propeller-twist, smaller slide and positive roll values, seen in AU/AU steps in RNA favors this cross-strand interaction, while the intra-strand N-H..O hydrogen bond is relatively infrequent. GC/GC step does not show significant occurrence of any intra-strand or cross-strand interaction between their exo-cyclic groups.RY steps in A-form helices are characterized by large intra-strand overlap and the exo-cyclic atoms in the major groove are positioned almost above each other, leading to unfavorable N-H..O angles, in both AU/AU and GC/GC steps. However, AC/GU step show presence of a weak intra-strand N-H..N interaction between the exo-cyclic amino groups in Adenine N6) and Cytosine (N4) . This type of hydrogen bond is observed in ~65% of CA/UG steps, , which can influence the dinucleotide step geometry. Roll values differentiate the three types of steps in RNA. RY steps have small roll (except for AU/AU steps), YR have high roll and RR have intermediate roll values. Interestingly, while roll values in DNA vary from small negative to small positive, a similar trend is seen with roll for YR > RR > RY steps. In B-DNA, the difference in parameters between RR, RY and YR is attributed to the effect of exocyclic groups on slide, roll and twist. Unlike the dinucleotide steps in B-DNA, which show a large variation in twist value that is strongly correlated with roll and moderately with slide, the twist values of steps in RNA helices cluster within a small range and show a significant correlation only with slide. The larger positive roll and negative slide values lead to a large number of favourable intra as well as cross-strand interactions involving the exo-cyclic groups, particularly in YR steps. Interestingly the slide values of WC steps show a significant negative correlation with average propeller-twist in RNA and a positive correlation for B-DNA. Steps with large average propeller-twist  have smaller negative slide values in RNA. The proximity of atoms on major groove side, arising due to high roll and negative propeller-twist prevents large negative slide. Thus, the slide parameter is directly influenced by propeller-twist of the constituent basepairs, but as the basepairs become near planar (average propeller twist ≈ 0°), slide becomes large negative in RNA and small positive in B-DNA steps. Overall, the six dinucleotide step parameters in RNA helices show mean values as well as correlated variations that are different from those observed in B-form DNA,6,19,49.cont) and non-contacting (non-cont) dataset, showed statistically significant difference in comparison to free-RNA dataset for roll, slide and average propeller-twist. This suggests that, apart from protein induced conformation change on direct contact, it can allosterically affect the steps that are not in direct physical contact.The specificity in RNA-protein interaction is thought to be mainly brought about by the exposed bases which arRecently, various additional hydrogen bond interactions have been reported from the RNA double helical regions, between the base and phosphate group oxygens (BPh),27,43. AAmong YR steps, CG/CG and CA/UG steps, in addition to intra-strand N-H..O and N-H..N hydrogen bonds, they also have an unusual N-H..N interaction between 2-amino groups of Guanines and N9 atoms of Purine bases in these dinucleotide steps. Hydrogen bonds are generally associated with the electronegative character of the donor and acceptor atoms. Electrostatic potential (ESP) derived charges, as well as partial charges in AMBER and CHARMM force fields assign near zero charges to the N9 atoms in Adenosine and Guanosine-54. HoweIt should also be mentioned that x-ray determined crystal structures do not have coordinates of hydrogen atoms. Programs that add hydrogen atoms to the nucleotide ring atoms as well as the N atoms in the pendent amino group, place the hydrogen atoms in the plane of the base, though many QM studies suggest that these amino groups can have pyramidal geometry with hydrogen atoms being out-of-plane,56-59. TApart from using the well-known Watson-Crick edge, a base can pair with other bases using the Hoogsteen or Sugar edges. In our crystal dataset we focused only on the dinucleotide steps formed by WC basepair that belong to the cisWW family (~74%), to compare with equivalent dinucleotide steps in DNA helices. However, more than 32 types of cisWW steps, containing at least one non-Watson-Crick basepair, such as GU, AG and UU, are also present and constitute ~17% of the total number of steps, while ~9% of the steps contain bases that are paired along the Hoogsteen or Sugar edge. Hence, to get a complete picture of the RNA helical geometries, the non-canonical basepair containing steps were analysed, but the small number present for each of these step types in crystal structures poses a challenge in arriving at any statistically significant result.Our analysis of the non-redundant RNA crystal structures shows that the RNA dinucleotide steps have characteristic sequence dependent variations. Overall, the steps show features that are attributes of their being of RR, RY or YR type. Several cross-strand and intra-strand potential hydrogen bonds are found to be highly prevalent in RNA helices and can be attributed to the observed geometrical preferences of various dinucleotide steps. Unusual cross-strand interactions are found to be present in the CA/UG and CG/CG steps, between 2-amino groups of Guanines and N9 atoms of Purine bases that are associated with the unique geometry of YR steps. The various dinucleotide steps in RNA bound to proteins show some significant differences in their dinucleotide parameters, from those in free RNA, while retaining most of the gross features, as well as the non-canonical cross-strand and intra-strand interactions.The authors declare that they have no competing interest.DB and MB conceived the project. SK carried out the analysis. All authors participated in the writing of the manuscript. All authors read and approved the final manuscript.Overall cross-correlation between dinucleotide step parameters for all steps comprising of canonical WC basepairs. For free-RNA dataset (N = 797): correlation coefficient (r) values ≥ 0.15 are significant at 99.9% confidence level. For bound-RNA dataset (N = 2531): correlation coefficients (r) values ≥ 0.10 are significant at 99.9% confidence level. For ADNA dataset (N = 195): correlation coefficients (r) values ≥ 0.32 are significant at 99.9%. For BDNA dataset (N = 212): correlation coefficients (r) values ≥ 0.23 are significant at 99.9% confidence level. ‘r’ values significant at 99.9% level, in each dataset, are shown in bold.Click here for fileDetails of the structures from which representative RR, RY and YR steps are taken in Figures 5,6, and7. The PDB IDs, residue ids, bases and atoms involved in intra-strand and cross-strand hydrogen bonding, as well as DA, HA distances and DHA angle are listed for each step. Distances marked in the figures are shown in bold.Click here for fileComparison of non Watson-Crick hydrogen bonds observed in all RNA and DNA helices. Mean and SD values of donor-acceptor distances and angles observed for cross-strand and intra-strand hydrogen bond in more than 50% of the 10 dinucleotide steps are listed. Hydrogen bond parameters in various model structures are also given.Click here for filePartial charges assigned to atoms in Adenine and Guanine nucleosides. The partial charges derived from Natural Bond Orbital (NBO) and Electrostatic potential (ESP) calculations are listed, along with those used in AMBER and CHARMM force fields. The large negative charge assigned by NBO calculation is highlighted in bold for the N9 atoms of adenine and guanine bases, which are involved in an unusual cross-strand hydrogen bond with the 2-amino group of guanine in the CA/UG and CG/CG steps respectively (shown in Figure Click here for file


O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus  specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′- O-methyladenosines are not active substrates for internal methylation, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis confirmed that the internal methylation product is 2′-O-methyladenosine. Furthermore, the 2′-O-methyladenosine could also be detected in DENV genomic RNA. The 2′-O methylation of internal adenosine does not require specific RNA sequence context because the DENV methyltransferase can methylate RNAs spanning different regions of viral genome and host ribosomal RNAs at equal efficiencies. Mutagenesis analysis showed that K61-D146-K181-E217 motif of the DENV methyltransferase forms the active site of internal methylation activity; in addition, distinct residues on the surface of the enzyme are critical for the internal methylation activity. Functional analysis showed that internal methylation attenuated viral RNA translation and replication. Overall, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNA in vitro. Such 2′-O-methyladenosine modification may modulate virus-host interaction.We report that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant proteins of NS5 and its N-terminal methyltransferase domain of West Nile virus and Dengue virus (DENV) specifically methylates polyA, but not polyG, polyC, or polyU. RNAs with internal adenosines substituted with 2′- Flavivirus genus from Flaviviridae family are important human pathogens, including the four serotypes of Dengue virus (DENV-1 to -4), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). These viruses are naturally transmitted by mosquitoes or ticks, causing global burden and threat to public health 7GpppAmG). The genomic RNA consists of a 5′ untranslated region (UTR), a single open-reading-frame, and a 3′ UTR. The open-reading-frame encodes a long polyprotein that is processed by viral and host proteases into three structural proteins  and seven nonstructural proteins  Many members within the O methylation activities required for the formation of 5′ RNA cap 7GpppA-RNA→m7GpppAm-RNA. Both reactions use S-adenosyl-L-methionine (SAM) as the methyl donor and generate S-adenosyl-L-homocysteine (SAH) as a by-product. The order of two sequential methylations is dictated by the fact that the 2′-O methylation reaction prefers the substrate m7GpppA-RNA to GpppA-RNA, whereas the N7 methylation reaction has no preference between substrates GpppA-RNA and GpppAm-RNA 7 and 2′-O methylations through an RNA cap-repositioning mechanism 7 methylation of flaviviral RNA cap is critical for efficient translation O methylation functions in subverting innate host antiviral response through escape of IFIT-mediated suppression We previously showed that the N-terminal domain of flaviviral NS5 protein posses both N-7 and 2′-6-methyladenosine (m6A) represents a major internal modified nucleoside. The m6A is found in cellular mRNAs from mammals, plants, insects, and yeast 6A modification functions in mRNA processing 6A, 2′-O methylation of ribose represents another common internal nucleoside modification. The 2′-O methylation is found in splicesomal small nuclear RNAs (snRNAs) and ribosomal RNAs O methylations remains elusive, these modifications are clustered in regions of functional importance, such as regions engaged in RNA-RNA interactions O methyl group could modulate RNA structure, thermal stability, biochemical interactions, and other aspects of the modified RNA Most eukaryotic mRNAs contain co- or post-transcriptional modifications, including the 5′ cap structure, internal bases methylation, splicing of introns, and polyadenylation. NO methylation occurs specifically at internal adenosine, not at guanosine, cytidine, or uridine. Mutagenesis analysis indicates that K61-D146-K181-E218 tetrad of the DENV-4 MTase forms the active site to catalyze internal methylation. Functional studies, using flavivirus luciferase replicon and genome-length RNAs, indicate that internal Am modification reduces viral RNA translation and RNA synthesis. Furthermore, we found that recombinant flavivirus NS5 can methylate host ribosomal RNAs in vitro.Here we report that flavivirus NS5 performs methylation at the 2′-OH position of internal adenosine (Am) of RNA. The 2′-in vitro transcribed in the presence of biotinylated CTP. The biotinylated pppA-RNA was incubated with DENV-4 MTase in the presence of [methyl-3H]-SAM. The methylation reaction was then incubated with SPA beads coated with streptavidin. If the RNA is methylated, binding of the biotinylated RNA to the streptavidin SPA beads brings the [methyl-3H]-labeling into close proximity to the scintillant (embedded in the beads), leading to a signal that can be measured by a scintillation counter. As shown in 3H]-signal upon treatment of DENV-4 MTase. In contrast, no 3H-activity was detected after the pppA-RNA was treated with DENV-4 RdRp domain. Addition of the RdRp domain to the MTase domain did not improve the methylation activity, whereas the full-length (FL) NS5 showed higher activity than the MTase domain alone. Interestingly, similar amounts of 3H-activity were detected after the WNV pppA-RNA and DENV-4 pppA-RNA were treated with DENV-4 FL NS5 and WNV FL NS5, respectively (data not shown). These results demonstrate that (i) DENV-4 MTase can methylate viral RNA without a 5′ cap structure; (ii) the RdRp domain could enhance the MTase activity, but only when the two domains are physically connected; and (iii) WNV and DENV-4 NS5 can cross methylate heterologous viral RNA.We developed a scintillation proximity assay (SPA) detect methylation of RNA without a 5′ cap structure . A pppA-O methylation occurs after the pppA-RNA was capped with a 5′ G ), we prepared a pppGGA-RNA that contained two extra G residues (underlined) to the 5′ end of authentic viral sequence. Methylation reactions showed that, compared with the pppA-RNA, the addition of two G residues did not change the methylation signals (data not shown). These results suggest that (i) the observed methylation activity is not dependent on the position of the first A residue; (ii) the 3H-signals could be derived from internal methylation of the RNAs without 5′ cap.To exclude the possibility that the observed methylation occurs at the first nucleotide A  and GpppA-RNA (to GpppAm-RNA); the methylation signals derived from m7GpppA-RNA were higher than those derived from GpppA-RNA , whereas it can only methylate one position on substrate m7GpppA-RNA (to m7GpppAm-RNA). Comparison of methylation signals showed that (i) the VP39-mediated 2′-O methylation is more robust than the flavivirus MTase-mediated cap methylations; and (ii) flavivirus MTase methylates RNA cap more efficiently than internal nucleoside. Taken together, these results indicate that flavivirus NS5 can methylate RNA without a cap structure, possibly through methylating internal nucleoside(s).We expanded the above observation to WNV, another member of flavivirus. Recombinant proteins of WNV FL NS5, MTase domain, RdRp domain were prepared. SPA analysis using pppA-RNA representing the first 190 nt of the WNV genome sequence showed that both FL NS5 and MTase domain, but not RdRp domain, could methylate the pppA-RNA . As a nesequence . In contpppA-RNA , confirmubstrate . This is2. Addition of MnCl2 inhibited the internal methylation activity.Using substrate pppA-RNA (5′ 211 nt of DENV genomic RNA) and DENV-4 MTase, we determined the optimal condition for internal methylation activity. As shown in 3H]-SAM. The purified RNAs were then measured for the level of 3H-methyl incorporation using a scintilation counter. The results showed that DENV-4 MTase efficiently methylated polyA . Since the homopolymer RNAs were not biotinylated, the methylation reactions were purified through an RNeasy column (Qiagen) to remove the un-incorporated -SAM (PerkinElmer) in buffer containing 50 mM Tris-HCl (pH 9.0) and 50 mM NaCl at room temperature for 1 h. The reactions were terminated by addition of an equal volume of 2× stop solution . The 96-well plate was agitated at room temperature for 15 min, and measured for 3H-methyl incorporation (into RNA) by a MicroBeta counter (Perkin-Elmer). The full-length NS5 and MTase domain from both WNV and DENV-4 were used in the methylation assays. The MTase domains of WNV and DENV-4 contained the first 300 and 272 amino acids of their respective NS5 proteins. The preparations of NS5 and MTase proteins were reported previously Three assays were performed to detect internal RNA methylation. The first assay used SPA format in a 96-well plate . Biotiny3H-methyl] incorporation into non-biotinylated RNA substrates. The reaction (20 µl) contained 50 mM Tris-HCl (pH 9.0), 2 µg MTase of DENV-4, 1 µCi of [3H-methyl]-SAM, and 1 µg of oligo RNA, viral RNA, 18 S rRNA, or 28 S rRNA. After incubation at room temperature for 1 h, the unincorporated [3H]-SAM was removed by RNeasy kit  according to the manufacture's instruction. The RNA samples were then mixed with 50 µl of optiphase supermix (Perkin Elmer), and measured for [3H-methyl] incorporation by a MicroBeta counter.The second assay measured [A third assay involved LC-MS analysis of Am following treatment of polyA, 18S rRNA and 28S rRNA (1 µg) with DENV-4 MTase (2 µg) in a reaction  containing 50 mM Tris-HCl (pH 9.0), 2 mM DTT and 50 µM SAM, with incubation at room temperature for 1.5 h. In addition, genomic RNA purified from DENV virion was directly analyzed using LC-MS (see below).For analysis of internal adenosine methylation of genomic RNA, WT and MTase E217A mutant DENV-1 virions, grown in mosquito C6/36 cells, were purified. Briefly, C6/36 were infected with DENV-1 at an MOI (multiplicity of infection) of 0.1 and incubated at 29°C for five days. Cell culture supernatants were then harvested and virus were precipitated using 8% PEG8000 (w/v) at 4°C overnight. Precipitated virus was then resuspended in NTE buffer  and purified by spinning the virus through a 24% (w/v) sucrose cushion at 75,350× g for 1.5 h at 4°C. Virus pellet was resuspended into 4% (w/v) potassium tartrate in NTE buffer and centrifuged at 149,008× g for 2 h at 4°C. Virus was further purified in a 10–30% potassium tartrate gradient by spinning at 126,444× g for 2 h at 4°C. Virus band of WT or E217A mutant was collected and concentrated using Millipore Amicon Ultra 100 K MWCO (Molecular Weight Cutoff). Virus samples were analyzed on a 15% SDS-PAGE stained with Coomassie brilliant blue to visualize viral capsid, membrane (pre-membrane), and envelope proteins. The amount of purified WT and E217A mutant virus were similar. Genomic RNAs were extracted from the purified virions using trizol (Invitrogen), quantified using NanoDrop, and subjected to enzymatic hydrolysis as described below.Identification and quantification of Am in samples of MTase-treated polyA, and 18S and 28S rRNA, and in DENV genomic RNA was achieved by analysis of RNA-derived ribonucleosides by HPLC-coupled mass spectrometry. For all analyses, samples of RNA (1–3 µg) were treated with 1 U/µl nuclease P1, 2.5 mM deferoxamine mesylate (antioxidant), 10 ng/ml coformycin (adenosine deaminase inhibitor), 50 µg/ml tetrahydrouridine (cytidine deaminase inhibitor), and 0.5 mM butylated hydroxytoluene (antioxidant) at 37°C. After 3 h, alkaline phosphatase and phosphodiesterase I were added to a final concentration of 0.1 U/ml. The sample was incubated at 37°C overnight, followed by removal of enzymes by filtration through a 10,000 kDa-molecular weight cut-off Amicon spin filter. The resulting filtrate was lyophilized prior to mass spectrometric analysis.m/z 100–1000) with the following parameters: gas temperature, 350°C; drying gas, 10 l/min; fragmentor voltage, 100 V; skimmer voltage, 65 V; and capillary voltage, 3500 V. Data processing was performed using MassHunter Workstation software version B.03.01 . The retention times and exact molecular weights of Am and other methylated ribonucleoside species were compared to chemical standards .For identification of Am in polyA treated with MTase, the lyophilized hydrolysis products were dissolved in deionized water and analyzed by HPLC-coupled, electrospray ionization (ESI) quadrupole time-of-flight mass spectrometry (LC-QTOF). To resolve ribonucleosides, the digested sample (5 µl) was loaded onto a Thermo Hypersil aQ column  at 25°C and eluted at a flow rate of 50 µl/min with an acetonitrile gradient using the following mobile phases: Solvent A: 0.1% formic acid in 10 mM ammonium acetate (pH 7.5); and Solvent B: 0.1% formic acid in acetonitrile. The percentage of Solvent B was as follows: 0–5 min, 0.5%; 5–14 min, 5%; 14–19 min, 5%; 19–31 min, 23%; 31–34 min, 23%; 34–49 min, 83%; 49–54 min, 83%; 54–60 min, 0.5%; 60–70 min, 0.5%. The eluent was analyzed on an Agilent QTOF 6520 mass spectrometer with an ESI source operated in positive ion mode and the mass spectrometer was operated in ion scanning mode  or adenosine (0.1–10 µM) dissolved in the hydrolysis buffer as a matrix control. The number of Am per viral genome was calculated by multiplying the measured value of Am per adenosine by the number adenosines in the 10,735 ribonucleotide DENV-1 genome .To quantify Am, samples of RNA  were hydrolyzed to ribonucleosides, as described above, followed by resolution of the ribonucleosides on a Thermo Hypersil aQ column  at 25°C at a flow rate of 0.3 ml/min with an acetonitrile gradient in 0.1% (v/v) formic acid in water as follows: 0–1.5 min, 0%; 1.5–2.7 min, 6%; 2.7–4.4 min, 17.8%; 4.4–5.0 min, 18%; 5.0–5.5 min, 18%; 5.5–8.1 min, 30%; 8.1–9.3 min, 40%; 9.3–10.2 min, 65%; 10.2–11.1 min, 95%; 11.1–12.0 min, 95%; 12.0–12.9 min, 0%; 12.9–15.0 min, 0%. The HPLC column was coupled to a triple quadrupole mass spectrometer (LC-MS/MS) operated in positive ion, multiple reaction monitoring (MRM) mode for the Am molecular transition of Renilla luciferase (Rluc)-reporter replicons of DENV-1  in vitro transcribed using mMESSAGE mMACHINE kits (Applied Biosystems). The 5′ end of replicon or genome-length RNA was treated with vaccinia virus capping enzyme and VP39 to generate m7GpppAm-RNA following the manufacturer's protocols (Epicetre). The reactions were extracted with phenol∶chloroform and precipitated using ethanol. The resulting replicon RNAs (4 µg) were treated with 2 µg of WT and mutant WNV or DENV-1 MTases in the presence of 50 µM SAM in the methylation buffer described above. After incubation at room temperature for 1 h, the reaction mixtures were directly electroporated to 8×106 BHK-21 cells 5 cells per well), and assayed for luciferase activities at indicated time points. The luciferase assay was performed as reported previously 5′-TGAAGCTAAGGTGCTTGAGC-3′ and reverse primer 5′-AGCCACATCTGGGCATAAGA-3′) and another primer pair targeting housekeeping gene actin (forward primer 5′-AGAGGGAAATTGTGCGTGAC-3′ and reverse primer 5′-CAATGGTGA TGACCTGGCCA-3′) The RT-PCR reactions were performed using SuperScript III one-step RT-PCR kit (Invitrogen), and the products were analyzed on a 1% agarose gel.Besides measuring luciferase activity, we also quantified intracellular viral RNA at various time points after electroporation. For each time point, total cellular RNA was extracted using RNeasy kit (Qiagen). The extracted RNAs (3 µg) were subjected to standard RT-PCR quantification using one primer pair targeting viral NS5 gene (forward primer in vitro transcribed from a full-length cDNA plasmid linearized by SacII7GpppAm cap (2 µg) was treated with WT and mutant DENV-1 MTases. The internally methylated genome-length RNAs were electroporated into BHK-21 cells 5 BHK-21 at 16–24 h before the assay day). After incubating the plates for 6 h , culture medium was aspirated and replaced with an overlayer medium . The plates were incubated at 37°C with 5% CO2 for 5 days. The cells were then fixed with 10% formaldehyde for 20 min at room temperature, rinsed with tap water, and stained with 1% crystal violet for 5 min. The plates were again rinsed with tap water (to remove staining) and visually counted for plaques. The specific infectivity was calculated as the number of infectious virus upon transfection of 1 µg of genome-length RNA.Genome-length RNA of DENV-1was 2, 2 mM MnCl2, 0.25 µM annealed RNA template/primer, 1 µM cold UTP, 1 µM 3H-UTP, 4 mM DTT, and 50 nM full-length DENV-4 NS5. After incubating the reaction at room temperature for 1 h, the reactions were terminated by addition of an equal volume of 2× stop solution . The 96-well plate was agitated at room temperature for 15 min, and measured for 3H-UTP incorporation as described above.The sequences of RNA template and primer are shown in