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Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non?Segmented Negative?Sense RNA VirusJianrong Li1, 2, 3 and Yu Zhang1[1] Department of Food Science and Technology, College of Food, Agricultural and Environmental Sciences, The Ohio State University, USA[2] Division of Environmental Health Sciences, College of Public Health, The Ohio State University, USA[3] Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA1. This intricate 3D model was done by Visual Science and it’s based on X-ray analysis and data that was published in the last twenty years.
Rhabdoviruses are of the Mononegavirales order, which include nonsegmented, single-stranded, negative-sense RNA viruses. Structure-based amino acid sequence alignments of conserved domain VI of representative NNS RNA virus L proteins with known 2'-O methyltransferase, the E. IntroductionThe non?segmented negative?sense (NNS) RNA viruses encompass a wide range of significant human, animal, and plant pathogens including several National Institute of Allergy and Infectious Diseases (NIAID) Category A and C biodefense pathogens.
Please check your inbox for an email with subject "Gadgets Matrix: Please Confirm Subscription". The Rhabdoviridae family includes the Lyssavirus , Ephemerovirus , and Vesiculovirus genera. The virion has a distinct bullet shape with a helical nucleocapsid and envelope with about 400 large trimeric glycoprotein spikes on the surface. Rhabdoviruses encode 5 proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L).
VSV encodes five structural proteins: nucleocapsid (N), phospho- (P), matrix (M), glyco- (G), and large (L) proteins. The rabies virus is a member of the lyssavirus genus, and vesicular stomatitis virus belongs to the vesiculovirus genus. Signature motifs for nucleotide polymerization, mRNA cap addition, and cap methylation are shown.4. First, pppNNNN-RNA is hydrolyzed by an RNA triphosphatase (RTPase) to yield the diphosphate ppNNNN-RNA.
Second, an RNA guanylyltransferase (GTase) transfers GMP to ppNNNN-RNA to yield GpppNNNN-RNA. The side chains of amino acid residues (Y22, F180, E233 and D182) involved in binding of 7mGp cap are shown as sticks. RSV, hMPV, and PIV3 account for more than 70% of acute viral respiratory diseases, especially in infants, children, and the elderly [1, 2].
G-N-7 methlyation may be facilitated by the contact with the RNA molecule remains bound to the polymerase at the end of the initial methylation at the 2'-O position.
The model was generated based on the previous predicted structure of VSV MTase (amino acid residue from 1644 to 1842 in L protein) using PyMOL software.
Or, methylation of RNA at the 2'-O position may induce a conformational change that facilitates the repositioning of the RNA for subsequent G-N-7 methylation. In addition, measles remains a major killer of children worldwide, despite successful vaccination programs in developed countries [4]. Predicted alpha-helical regions are shown by the cylinders and the ?-sheet regions by the arrows.
The most virulent strains of NDV, the viscerotropic velogenic strains (often called “exotic” NDV), are classified as High Consequence Livestock Pathogens by USDA due to their potential as agents of agricultural bioterrorism [5]. SAM and the side chains of critical residues are shown in sticks, and the oxygen atoms and nitrogen atoms are shown in red and blue, respectively.
Messenger RNA modification is the essential issue in NNS RNA virus gene expression and replication. During viral RNA synthesis, NNS RNA viruses produce capped, methylated, and polyadenylated mRNAs [6-8].
For the three important amino acids (Y1650, F1691 and E1764) that may be involved in RNA substrate binding, their side-chain carbon atoms are highlighted in orange. Cap formation is essential for mRNA stability, efficient translation, and gene expression [9-11]. It is now firmly established that mRNA capping and methylation in NNS RNA viruses evolves in a mechanism distinct to their hosts [12-19]. For decades, VSV has been used as a model to understand the replication and gene expression of NNS RNA viruses. Second, a polyribonucelotidyltransferase (PRNTase) (CR V of L protein) transfers a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate to GpppAACAG-RNA. Most of our understanding of mRNA modifications of NNS RNA viruses comes from studies of VSV, a prototype of the Rhabdoviridae family. Third, the cap structure is methylated a 2’-O MTase (CR VI of L protein) to yield GpppAmACAG-RNA. The structure of VSV virionsVSV virions are bullet-shaped particles 170 nm in length and 80 nm in diameter (Fig.1).
Among NNS RNA viruses, VSV has the simplest RNA genome consisting of 11,161 nucleotides (nt) organized into five VSV genes encoding nucleocapsid (N), phospho- (P), matrix (M), glyco- (G), and large (L) proteins, and leader and trailer regulatory sequences arranged in the order 3’-(leader), N, P, M, G, L, (trailer)-5’[20-23]. Like all NNS RNA viruses, the genome is encapsidated with the N protein to form a nuclease-resistant helical N-RNA complex that is the functional template for mRNA synthesis as well as genomic RNA replication. This structure contains the minimum virus encoded components of the VSV RNA synthesis machinery [26]. The RNP complex is further surrounded by the M protein which plays a crucial role in virus assembly, budding, and maintenance of the structural integrity of the virus particle [27]. Upon attaching to an unknown cell receptor(s), VSV enters host cells via receptor mediated endocytosis [29]. Following low pH triggered fusion and uncoating, the RNP complex is delivered into the cytoplasm where RNA synthesis and viral replication occur [30]. During primary transcription, the input RdRp recognizes the specific signals in the N-RNA template to transcribe six discrete RNAs: a 47-nucleotide leader RNA (Le+), which is neither capped nor polyadenylated, and 5 mRNAs that are capped and methylated at the 5’ end and polyadenylated at the 3’end.
These mature mRNAs are then translated by host ribosomes to yield functional viral proteins which are required for viral genome replication. During replication, the RdRP initiates at the extreme 3’ end of the genome and synthesizes a full-length complementary antigenome, which subsequently serves as template for synthesis of full-length progeny genomes.
These progeny genomes can then be utilized as templates for secondary transcription, or assembled into infectious particles.
In response to a specific promoter element provided by the genomic leader region, the polymerase initiates mRNA synthesis at the first gene-start sequence to synthesize N gene. The nascent mRNA is capped through an unconventional mechanism in which the GDP: polyribonucleotidyltransferase (PRNTase) of L transfers a monophosphate RNA onto a GDP acceptor through a covalent protein-RNA intermediate [12, 13, 16].
Following cap addition, VSV mRNAs are sequentially methylated at ribose 2’-O position and G-N-7 position, which is distinct from all known methylation reactions [17, 18]. Unlike traditional cap forming enzymes, the VSV capping and methylation machinery requires cis-acting signals in the RNA [12, 18, 31, 32].
When encountering a gene-end sequence, L polyadenylates and terminates mRNA synthesis by a programmed stuttering of the polymerase on a U7 tract [33, 34]. Termination at the end of the N gene is essential for the polymerase to initiate synthesis at the start of the next gene, to produce the P mRNA. During transcription, the RdRp complex transcribes the viral genome into five mRNAs in a sequential and gradient manner, such that 3? proximal genes are transcribed more abundantly than 3? distal genes [21-23].
This gradient of transcription reflects a poorly understood transcriptional attenuation event that is localized to the gene junction regions.
Using this fashion, VSV produces five capped, methylated, and polyadenylated mRNAs, N, P, M, G, and L. Large (L) polymerase protein, the multifunctional protein that modifies viral mRNAAll NNS RNA viruses encode a large (L) polymerase protein, a multifunctional protein ranging from 220-250kDa in molecular weight. The L protein contains enzymatic activities for nucleotide polymerization, mRNA cap addition, cap methylation, and polyadenylation. To date, the structure of L protein, or L protein fragments, has not been determined for any of the NNS RNA viruses. Amino acid sequence alignment between the L proteins of representative members of each family within NNS RNA viruses has identified six conserved regions numbered I to VI (CRs I–VI) (Fig.3) [35]. Thus, there is a general assent that the enzymatic activities of L protein are located in these conserved regions. For the last four decades, VSV L protein has been used a model to understand the different activities of NNS RNA virus L proteins because it is the only member of this order of viruses for which robust transcription can be reconstituted in vitro [6, 7, 21, 36].
In addition, the VSV L protein can be highly expressed in recombinant expression systems, such as E.coli and insect cells. The purified VSV L protein retains all the enzymatic activities that can modify short virus-specific mRNA in trans [12, 16, 18, 37]. In recent years, many breakthroughs have been made in the characterization of the function of VSV L protein and the enzymatic activities have been mapped at the single amino acid level (Fig.3).
Within the primary sequence of L are six conserved regions shared among all NNS RNA virus L proteins. The RdRP activity has been identified in CR III and this region is also required for polyadenylation [38-40].
Consistent with this, a GDN motif is conserved in CR III of all NNS RNA virus L proteins and is functionally equivalent to the GDD polymerization motif characteristic of positive strand RdRPs. The mRNA capping enzyme maps to CR V [13, 16], and the capping activities of L differ from those of other viruses and their eukaryotic hosts.

Specifically, an RNA:GDP PRNTase activity present within CR V transfers 5? monophosphate RNA onto a GDP acceptor through a covalent L-pRNA intermediate [12, 13, 16]. Like the unconventional capping enzyme, methylation of the VSV mRNA cap structure is also unique in that mRNA cap is modified by a dual specificity MTase activity within CR VI whereby ribose 2?-O methylation precedes and facilitates subsequent guanine-N-7 (G-N-7) methylation [17, 18]. Signature motifs for nucleotide polymerization, mRNA cap addition, and cap methylation are shown.The location of the nucleotide polymerization, capping, and cap methylation activities within separate regions of L has led to the notion that L protein may be organized as a series of independent structural domains. In addition, recombinant VSV and measles virus can be recovered from infectious cDNA clones by inserting the coding sequence of green fluorescent protein between CR V and VI in L gene, suggesting that L protein folds and functions as a series of independent globular domains [44, 45].
Interestingly, mutations to a variable region between CRs V and VI (residues 1450–1481) affect mRNA cap MTase activity, feasibly suggesting that mutation to this hinge region may affect a conformational change in CR VI [46]. More recently, the molecular architecture of VSV L protein has been revealed using negative stain electron microscopy (EM) in combination with proteolytic digestion and deletion mapping [37]. It was found that VSV L protein is organized into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Interestingly, upon binding to P protein, L protein undergoes a significant structural rearrangement that may facilitate the coordination between mRNA synthesis and capping apparatus [37, 47].4.
Second, an RNA guanylyltransferase (GTase) reacts with GTP to form a covalent enzyme-GMP intermediate and transfers GMP to 5’ppN-RNA via a 5?-5? triphosphate linkage to yield 5’ GpppN-RNA. This mRNA capping reaction is conserved among all eukaryotes.Viruses are highly diverse in capping their mRNA. Many DNA viruses (such as vaccinia virus and baculovirus), double stranded RNA viruses (such as reovirus, rotavirus, and bluetongue virus), and single strand positive RNA viruses (such as West Nile virus, Fig.4B) utilize the conventional eukaryotic capping pathway [51-56]. An unconventional mRNA capping mechanism in VSVIn the early 1970’s, it was suggested that the cap structure of NNS viral mRNAs was formed by a mechanism which was unique from eukaryotic cap formation. For VSV [6], RSV [61], and spring viremia of carp virus [62], the two italicized phosphates of the 5?Gppp5?NpNpN triphosphate bridge have been shown to be derived from a GDP donor, rather than GMP.
However, further studies on this mechanism have been seriously hampered due to the fact that the VSV capping events are tightly coupled to transcription and the capping machinery does not respond to exogenous transcripts. In 2007, this unique capping mechanism was revealed using a novel trans capping assay, in which a short mRNA corresponding to the first 5-nt of VSV gene start sequence was capped by a highly purified L protein in trans [12]. Specifically, capping of VSV mRNA was achieved by a novel polyribonucelotidyltransferase (PRNTase) which transferred a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate (Fig. In the first step, a GTPase associated with the VSV L protein removes the ?-phosphate group of GTP to generate GDP, an RNA acceptor.
Interestingly, this unusual VSV capping enzyme caps RNA in a sequence specific manner [12].
The only suggestive information regarding the location of the capping enzyme in L protein has come from the study of a novel inhibitor of the RSV polymerase which resulted in the synthesis of short uncapped viral RNAs in vitro [63].
Viral mutants resistant to this inhibitor were selected, and the resistance mutations were mapped to CR V, suggesting that CR V of L plays a role in mRNA cap formation. Sequence alignments of this region of L protein identified a total of 17 residues that were conserved among the NNS RNA viruses [16].
In vitro RNA reconstitution assays have shown that these cap defective polymerases synthesized uncapped mRNAs that terminated prematurely. The size of these abortive transcripts ranged from 100 nt up to the full-length N mRNA, although the majority were less than 400 nt. Importantly, GxxT[n]HR is highly conserved in the CR V of L proteins of all NNS RNA viruses, including Borna disease virus which replicates in nucleus. Further biochemical and mass spectrometric analyses found that H1227 in the conserved GxxT[n]HR motif of the VSV L protein is covalently linked to the 5?-monophosphate end of the RNA through a phosphoamide bond [13]. Interestingly, this PRNTase activity was also found in L protein of Chandipura virus (CHPV), a rhabdovirus that is closely related to VSV [64].
Furthermore, mutations to HR motif in L protein of CHPV significantly reduced the formation of the L-pRNA covalent intermediates in the PRNTase reaction. These results demonstrate that this unconventional capping mechanism is conserved in the Rhabdoviridae family.
Given the fact the HR motif is highly conserved in L proteins of NNS RNA viruses, it is likely that this novel capping mechanism is not only unique to rhadoviruses, but also may be utilized by other NNS RNA viruses.5. First, the capping guanylate is methylated by a G-N-7 methyltransferase (MTase) to yield 7mGpppN-RNA (cap 0). Second, the G-N-7 methylated cap structure can then be further methylated by a ribose-2’-O (2’-O) MTase to yield 7mGpppNm-RNA (cap 1). During mRNA cap methylation, S-adenosyl-L-methionine (SAM) serves as the methyl donor, and the by-product S-adenosyl-homocysteine (SAH) is the competitive inhibitor of the SAM-dependent MTase. In this conventional methylation reaction, G-N-7 methylation occurs prior to 2’-O methylation and the two methylase activities are carried out by two separate enzymes, each containing its own binding site for the methyl donor, SAM. Many viruses encode their own mRNA cap methylaion machinery, the best-studied example of which is the poxvirus vaccinia virus. For vaccinia virus, the G-N-7 and 2’-O MTase activities are encoded by two separate viral proteins, D12L and VP39 [65, 68-70]. In the case of reovirus, G-N-7 and 2’-O MTases are catalyzed by two separate domains of the same viral polymerase protein [55, 71]. For VSV, G-N-7 and 2’-O MTases are accomplished by a single region (CR VI) located in the C terminus of viral polymerase protein, L (Fig.4C) [14, 17, 19]. Soon after the discovery of the dual MTase activities of VSV, the N terminus of flaviviruses polymerase protein (NS5) was found to encode both G-N-7 and 2’-O MTases (Fig.4B) [72-74].
In addition to this unusual dual MTase activity of CR VI, the order of mRNA cap methylation in VSV is unconventional in which 2’-O methylation precedes and facilitates the G-N-7 methylation [17, 18]. A single MTase catalytic site in CR-VI of L protein essential for both G-N-7 and 2’-O methylationThe SAM-dependent MTase superfamily contains a series of conserved motifs (X and I to VIII) [75].
In RrmJ, a catalytic tetrad of residues: K38, D124, K164, and E199 formed the active site of 2’-O MTase [67, 76]. Site-directed mutagenesis of RrmJ found that a catalytic triad of residues K38, D124, and K164 are essential for 2’-O MTase whereas E199 plays only a minor role in the methyltransferase reaction in vitro. In vaccinia virus VP39, four amino acids, K41, D138, K175, and E207, are essential for catalysis [68, 70].
By comparing the amino acid sequence of the RrmJ and VP39 with CR VI of the L protein of NNS RNA viruses, it was suggested that this region of L protein might function as a 2?-O MTase.
Sequence alignments suggest that residues K1651, D1762, K1795, and E1833 of the VSV L protein correspond to a catalytic KDKE tetrad (Fig.5). In fact, this KDKE motif is conserved in CR VI of L proteins of all NNS RNA viruses with the exception of Borna disease virus.
Li et al., (2005) performed an extensive mutagenesis analysis in this predicted MTase catalytic KDKE tetrad in VSV L protein [14]. Recombinant VSVs carrying individual substitutions to K1651, D1762, K1795, and E1833 were recovered from an infectious cDNA clone of VSV. Analysis of the cap structure of mRNA synthesized in vitro revealed that alterations to the predicted active site residues abolished both G-N-7- and ribose 2?-O MTase activities. This result demonstrated that a single KDKE tetrad in CR-VI of the VSV L protein is essential for mRNA cap G-N-7- and ribose 2?-O methylation [14]. However, this conflict the fact that all known G-N-7 and 2’-O MTases have distinct biochemistry during RNA methylation reactions. An alternative explanation is that there is a sequential model for VSV mRNA cap methylation in which the product of one MTase acts as the substrate for the second (discussed below).
Figure 5.Structure-based amino acid sequence alignments of conserved domain VI of representative NNS RNA virus L proteins with known 2'-O methyltransferase, the E. Sequence alignments between CR VI of NNS RNA virus L proteins and known MTases suggest that the SAM-binding residues of VSV L include G1670, G1672, G1674, G1675, and D1735 (Fig.5). Site-directed mutagenesis has been performed to define the roles of these amino acids in VSV mRNA methylation [17].
In addition, the flanking amino acid residues D1671 and S1673 within GDGSG motif were also substituted. It was found that mutations to G1670, G1672, and S1673 specifically diminished G-N-7, but not 2’-O methylation, suggesting that 2’-O methylation occurs prior to G-N-7 methylation in VSV [17]. Interestingly, mutant G1674A requires a higher concentration of SAM to achieve full methylation compared with wild type VSV and methylation is more sensitive to SAH inhibition. Therefore, amino acid substitutions to the predicted SAM binding site disrupted methylation at the G-N-7 position or at both the G-N-7 and ribose 2?-O positions of the mRNA cap. Mapping the potential RNA binding site that required for mRNA cap methylationTo acquire methylation, the MTase usually directly or indirectly contacts an RNA substrate. However, this substrate binding site has been identified in several cellular and viral mRNA 2'-O MTases [67, 68, 70, 74, 77, 78]. To achieve 2'-O methylation, the RNA substrate interacts with the cap recognition site which requires stacking between the base of the cap and aromatic rings from a MTase [76, 79, 80]. In VP39, it was found that the recognition of a methylated base is achieved by stacking between two aromatic residues (Y22 and F180) and the methyl group is in contact with residue Y204 (Fig.6A) [68, 70, 79]. In addition, the carboxyl groups of residues D182 and E233 form hydrogen bonds with the NH and NH2 of the guanosine in VP39 (Fig.6A).
Based on structure modeling and mutagenesis analysis, it was shown that residue F24 in West Nile virus (WNV) methylase (NS5) [81, 82] and Y29 and F173 in feline coronavirus 2'-O MTase (nsp16) [80] may play an equivalent role to residue Y22 in VP39 of vaccinia virus.

For the predicted catalytic residues (K1651, D1762, K1795 and E1833), the carbon atoms are shown in purple.Guided by this information, the putative RNA binding site in VSV L protein was searched through mutagenesis analysis of selected conserved residues in region VI of VSV L protein that were physiochemically similar to those involved in substrate recognition in VP39. Sequence alignment showed that there are a number of aromatic residues that are highly conserved in the MTase domain of L proteins of NNS RNA viruses (Fig. Aromatic residues at positions 1650 (Y), 1691(F or Y), and 1835 (Y) are highly conserved in L proteins of NNS RNA viruses. Aromatic residues at positions 1742 (W), 1744 (Y), 1745 (F), and 1816 (F) are conserved in the L proteins of Rhabdoviridae and some Paramyxoviridae and Filoviridae.
Therefore, these aromatic residues were selected as putative equivalents of Y22, F180, and Y204 in VP39. With the exception of two acidic amino acids in the catalytic site (K1651-D1762-K1795-E1833), position E1764 is also conserved in all L proteins. Thus, E1764 was selected as a candidate for mimicking the role of VP39 residues D182 and E233. In addition, two serine mutations at the two most conserved positions at 1693 and 1827 of VSV L protein was also examined, based on the fact that it has been shown that a serine residue was involved in RNA-protein interaction in E. To determine the role of these amino acid residues in mRNA cap methylation, a single point mutation was introduced to an infectious clone of VSV and recombinant VSVs harboring these mutations were recovered [84]. The importance of the maintenance of the aromatic ring at amino acids Y1650 and F1691 was revealed by the observation that the substitution of Y1650 and F1691 with two other possible aromatic residues in the VSV infectious clone still produced viable recombinant viruses and produced a fully methylated mRNA cap, but alanine substitutions dramatically inhibited viral replication and completely blocked both G-N-7 and 2’-O methylation [84]. Y1650 is located in the middle of the first helix, and the F1691 is at the very C-terminal of the second helix. Perhaps, a stacking interaction with one aromatic residue causes a conformational and structural change in the VSV methylase, which results in the interaction with another aromatic residue. Changing of residue E1764 to D (maintenance of charge), Q (maintenance of size), or K (changing charge), even the very conservative change to D, dramatically inhibited both G-N-7 and 2'-O methylation [84].
The predicted structure of VSV MTase also shows that E1764, the residue adjacent to the catalytic residue D1762, is exposed to the putative SAM binding site (Fig.6B). In addition, it was found that Y1835A was found to require a higher SAM concentration to achieve full methylation and it is more sensitive to MTase inhibitor [84].
To date, this work is the first attempt toward elucidation of the putative RNA substrate recognition site in the L protein of NNS RNA viruses, which has shed light on the possible role of several conserved aromatic amino acids, including Y1650 and F1691, in RNA binding during cap methylation.
It would provide much more direct evidence for the role of these key amino acids in mediating RNA binding if the RNA binding efficiency could be measured directly. Attempts to use a gel shift assay have failed to this end [84], as the existence of multiple RNA binding sites in L protein with a size as large as 241-kDa posed a tremendous challenge in discerning the effect of single point mutation. The use of a truncated CR VI of VSV L for in vitro RNA binding assays might be a useful alternative strategy for future studies.
An unusual order for mRNA cap methylation in VSVFor conventional mRNA cap methylation, two separate MTases sequentially methylated the cap structure, first at the G-N-7 position and subsequently at the ribose 2?-O position [65, 66].
Analysis of the cap methylation of mRNA synthesized in vitro suggests that mRNA cap methylation in VSV is unusual, with methylation of ribose 2?-O occurring prior to G-N-7 methylation.
First, early studies showed that at low concentrations of SAM, VSV mRNA was methylated at the 2’-O position only [85]. However, it could be chased into a doubly methylated cap structure at high SAM concentrations in vitro. Second, when in vitro mRNA synthesis was performed in the presence of MTase inhibitors such as SAH and sinefungin, G-N-7 methylation was inhibited prior to 2’-O methylation [86]. Third, a host range mutant of VSV, hr8, was shown to synthesize mRNA cap structures that lacked G-N-7 but were partially 2?-O-methylated [46, 87]. Finally, VSV mutants carrying mutations in the SAM binding site (such as G1670A, and G1672A) are specifically defective in G-N-7, but not 2’-O methylation [17]. This unusual order of VSV mRNA cap methylation was also biochemically demonstrated by a trans-methylation assay in which both ribose 2?-O and G-N-7 MTases were recapitulated by using purified recombinant L and in vitro-synthesized RNA [18].
It was found that VSV L modifies the 2?-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2?-O methylation of the substrate RNA [18], providing compelling evidence that 2?-O methylation precedes and facilitates G-N-7 methylation. Bearing in mind that G-N-7 position is upstream of the ribose 2'-O position in the mRNA strand, reorientation is thus less likely to have resulted from forward movement of capped RNA through CR VI during transcription, but rather it might entail a fine spatial rearrangement. Collectively, these experiments have shown that the order of VSV mRNA cap methylation is distinct from all other known mRNA cap methylation mechanisms. VSV methylases require cis-element in RNADuring mRNA synthesis, the VSV polymerase initiates synthesis at the first gene-start (GS) sequence (3? UUGUCNNUAC 5?), and the nascent mRNA chain is capped and methylated, and recognizes a specific gene-end (GE) sequence (3?-AUACUUUUUUU-5?), the polymerase polyadenylates and terminates. It has been well demonstrated that the GS sequence contains a key cis-acting regulatory element for the initiation of mRNA synthesis [31, 32].
Specifically, the first three positions of the GS sequence have been found to be critical for mRNA synthesis. Recently, both trans capping assays with 5-nt oligo RNA substrates and detergent-activated virus transcription reactions pointed out the importance of positions 1, 2, 3, and 5 in mRNA cap addition, although position 5 substitutions were more tolerated [12, 31, 32]. Using a trans methylation assay, it was found that similar signals were required for mRNA cap methylation [18].
As expected, VSV L protein efficiently methylated a 110 nt of RNA with an authentic gene start sequence at position 2’-O. However, when the gene start sequence of this 110 nt was replaced with non-viral sequence (5? GpppGGACGAAGAC-RNA), the efficiency of 2’-O methylation was reduced approximately 9 times.
Similarly, VSV L protein efficiently methylated a pre-2?-O-methylated VSV mRNA at position G-N-7. In contrast, the efficiency of G-N-7 methylation decreased nearly 7 times when incubated with a substrate with non-VSV mRNA start RNA. In order to determine the minimum length of RNA required for mRNA cap methylation, 5-,10-, 51-, and 110-nt RNAs were used as substrates for a trans methylation assay in vitro [18]. Interestingly, the 10-, 51-, and 110-nt RNAs were able to serve as substrates for both G-N-7 and ribose 2'-O methylations, whereas the 5-nt RNA was not methylated by the VSV L protein at either the G-N-7 or the ribose 2'-O position [18]. Therefore, in contrast to trans capping, a 5-nt substrate is not sufficient for trans methylation and likely the conserved positions 8, 9, and 10 in VSV gene start sequence are required for mRNA cap methylation. Clearly, the length of RNA required for methylation is longer than that required for capping by the VSV L protein.
Model for mRNA cap methylationThe process of VSV L protein-mediated cap methylation can be best summarized with the following model (Fig.7). Initially in response to a specific cis-acting element in the VSV gene start sequence, CR VI of L protein methylates the cap structure first at the 2'-O position to produce GpppAmACAG-RNA.
Following 2’-O methylation, a second molecule of SAM binds to CR VI of L protein that may facilitate a subsequent methylation of the RNA at the G-N-7 position. Methylation at the 2'-O position favors G-N-7 methylation in the cap structure through a currently unknown mechanism. G-N-7 methlyation may be facilitated by the contact with the RNA molecule remains bound to the L protein at the end of the initial methylation at the 2'-O position.
Comparison of mRNA cap methylation in VSV and WNVTo date, the rhabdovirus, VSV, and the flavivirus, WNV, are the two best characterized viruses that utilize a single region in the polymerase protein for both G-N-7 and 2'-O methylations. However, the mechanism of VSV mRNA methylation is distinct from that of the WNV system (Fig.4B and C).
However, WNV MTases modify the cap structure, first at the G-N-7 position and subsequently at the ribose 2'-O position [72, 73, 78].
In VSV, the G-N-7 and 2'-O MTases require similar conditions for methylation with an optimal pH at 7.0 [18]. In contrast, the G-N-7 and 2'-O MTases of WNV require an optimal pH at 6.5 and 10, respectively [72]. Both VSV and WNV MTases modify the RNA in a sequence-specific manner, but require different elements in the RNA substrate. VSV G-N-7 and 2'-O MTases require specific gene start sequences with a minimum mRNA length of 10 nucleotides [18].
In the WNV model, N-7 cap methylation requires the presence of specific nucleotides at the second and third positions and a 5' stem-loop structure within the 74-nucleotide viral RNA; in contrast, 2'-O ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides in length [81]. In addition, there is striking difference in the cap recognition site between the VSV and WNV MTases.
For the WNV MTase, the cap recognition site is essential for 2'-O, but not G-N-7 methylation [73, 82].
Consistent with this finding, it was found that GTP and cap analogs specifically inhibited 2'-O, but not G-N-7 methylation [73, 82].
However, mutations to the putative RNA binding site in VSV L protein affected both G-N-7 and 2’-O methylations. Overall, the mechanism of VSV mRNA cap methylation is significantly different from that of WNV. Most recently, it was found that capping of flavivirus RNA is catalyzed by conventional RNA guanylyltransferase via a covalent GMP-enzyme intermediate [88].

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