The 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. The NNS RNA viruses are classified into four families: the
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]. 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]. Thus, mRNA cap formation is an attractive antiviral target for NNS RNA viruses. For decades, VSV has been used as a model to understand the replication and gene expression of NNS RNA viruses. Most of our understanding of mRNA modifications of NNS RNA viruses comes from studies of VSV, a prototype of the
2. Overview diagram of VSV mRNA synthesis and modifications
2.1. The structure of VSV virions
VSV 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. The N-RNA complex is tightly associated with the viral RNA-dependent RNA polymerase (RdRp), which is comprised of the 241-kDa L protein catalytic subunit and the 29-kDa essential P protein cofactor, and results in the assembly of a viral ribonucleoprotein (RNP) complex [24, 25]. This structure contains the minimum virus encoded components of the VSV RNA synthesis machinery . 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 . The outer membrane of virion is the envelope composed of a cellular lipid bilayer. The transmembrane G protein is anchored in the viral envelope, which is essential for receptor binding and cell entry .
2.2. VSV life cycle
The overview picture of VSV life cycle is depicted in Fig.2. Upon attaching to an unknown cell receptor(s), VSV enters host cells via receptor mediated endocytosis . Following low pH triggered fusion and uncoating, the RNP complex is delivered into the cytoplasm where RNA synthesis and viral replication occur . 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. Finally, viral proteins and genomic RNA are assembled into complete virus particles and the virus exits the cell by budding through the plasma membrane.
2.3. VSV mRNA synthesis and modifications
Our current understanding of VSV mRNA synthesis and modification can be summarized as follows. 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
3. Large (L) polymerase protein, the multifunctional protein that modifies viral mRNA
All 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) . 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
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. Consistent with this idea, a fragment containing CRs V and VI of the SeV L protein were expressed independently and shown to retain the ability to methylate short RNAs that corresponded to the 5′ end of SeV mRNA . 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 . 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 . 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. An unconventional mRNA capping mechanism in VSV
4.1. Conventional mechanism of mRNA capping in eukaryotic cells
In eukaryotic cells, capping of mRNA is an early posttranscriptional event that is essential for subsequent processing, nuclear export, stability, and translation of mRNA [11, 48]. Cap formation is mediated by a series of enzymatic reactions (Fig.4A). First, the 5’ triphosphate end of the nascent mRNA chain (5’pppN-RNA) is hydrolyzed by an RNA triphosphatase (RTPase) to yield the diphosphate 5’ ppN-RNA. 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. Typically, RNA GTases contain a signature Kx[D/N]G motif that functions as an active site for the capping reaction [11, 48, 49]. A lysine residue within Kx[D/N]G motif forms the enzyme-GMP covalent intermediate, prior to its transfer onto the diphosphate RNA acceptor [11, 48, 50]. 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]. It has been suggested that Kx[V/L/I]S motif serve as the GTase active site for reovirus and rotavirus . Other viruses have evolved different mechanisms for acquiring their cap. For example, influenza virus and hantavirus furnish their mRNA with this structure by a cap-snatching mechanism, in which the viral polymerase steals host cell mRNA caps to prime viral mRNA synthesis [58, 59]. The alphaviruses, such as Sindbis, have evolved S-adenosyl-L-methionine(SAM
4.2. An unconventional mRNA capping mechanism in VSV
In 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 , RSV , and spring viremia of carp virus , the two italicized phosphates of the 5′G
It has been a challenge to locate the active site for the novel PRNTase in the 241 kDa L protein. 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
5. An unusual mechanism of mRNA cap methylation in VSV
5.1. Conventional mRNA cap methylation in eukaryotic cells
In eukaryotic cells, the capped mRNA (GpppN-RNA) is typically methylated by two steps (Fig.4A) [65-68]. 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. These mRNA cap methylation reactions are conserved among all eukaryotes. 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]. This is contrast to all known mRNA cap methylation reactions including flaviviruses.
5.2. A single MTase catalytic site in CR-VI of L protein essential for both G-N-7 and 2’-O methylation
The SAM-dependent MTase superfamily contains a series of conserved motifs (X and I to VIII) . The crystal structure of several known 2’-O MTases including
5.3. A single SAM binding site in L protein essential for both G-N-7 and 2’-O methylation
The SAM-dependent MTase superfamily usually contains a G-rich motif (GxGxG) and an acidic residue (D/E) that is involved in SAM binding . 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 . Each of these residues was individually substituted for alanine (A); or, for G4A, all four G residues were replaced with A; for G4AD, residue D1735 was also replaced with A. In addition, the flanking amino acid residues D1671 and S1673 within GDGSG motif were also substituted. Recombinant viruses were recovered from each of the
5.4. Mapping the potential RNA binding site that required for mRNA cap methylation
To acquire methylation, the MTase usually directly or indirectly contacts an RNA substrate. This putative substrate binding site is poorly understood in NNS RNA viruses. 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]. Vaccinia VP39 is one of the best characterized 2'-O MTases. 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)  may play an equivalent role to residue Y22 in VP39 of vaccinia virus. The cellular cap binding protein-eukaryotic translation initiation factor 4E (eIF-4E) recognizes the cap by stacking between W56 and W102 . In all known cases, aromatic residues are involved in cap binding and substrate recognition.
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. 5). 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
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 , 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.
5.5. An unusual order for mRNA cap methylation in VSV
For 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
This unusual order of VSV mRNA cap methylation was also biochemically demonstrated by a
5.6. VSV methylases require
cis-element in RNA
During 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
5.7. The length of mRNA in cap methylation
5.8. Model for mRNA cap methylation
The process of VSV L protein-mediated cap methylation can be best summarized with the following model (Fig.7). Initially in response to a specific
5.9. Comparison of mRNA cap methylation in VSV and WNV
To 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). In VSV, 2'-O methylation precedes and facilitates subsequent G-N-7 methylation [17, 18]. 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 . In contrast, the G-N-7 and 2'-O MTases of WNV require an optimal pH at 6.5 and 10, respectively . 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 . 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 . 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. GTP and cap analogs did not affect VSV mRNA cap methylation
5.10. mRNA cap methylation in other NNS RNA viruses
Limited accomplishments have been made in understanding the mechanism of mRNA cap methylation in other NNS RNA viruses, due to the lack of a robust
SS(+) RNA: single strand positive RNA viruses include DNV, dengue virus; WNV, West Nile virus; YFV, yellow fever virus.
6. The effects of 5’ mRNA cap addition and cap methylation on 3’ mRNA polyadenylation
VSV mRNA is capped and methylated at the 5′ end and polyadenylated at the 3′ end. Cap addition, cap methylation, and polyadenylation are carried out by three different regions (CR V, CR VI, and CR III) in L protein. During VSV mRNA synthesis, modifications of the 5′ and 3′ ends of the mRNAs are tightly coupled to transcription [21-23]. Although the detailed mechanism by which the polymerase coordinates these modification events is poorly understand, available evidence suggests there is a link between authentic 5′-end formation and 3′-end formation during VSV mRNA synthesis. Early studies demonstrated that the length of poly (A) tails on VSV mRNAs is affected by the presence of SAH, the by-product and competitive inhibitor of SAM-mediated methyltransferases [93-95]. The fact that the polymerase can synthesize full-length mRNAs
The characterization of a panel of MTase-defective VSVs may serve as a tool to understand the mechanism by which SAH or the failure to methylate the cap structure results in hyperpolyadenylation. It was found that rVSV-K1651A, a mutation in MTase active site and completely defective in G-N-7 and 2’-O methylation, synthesized excessively long poly(A) tails, similar to those produced by wild-type L in the presence of SAH . Similarly, the substitution D1762E at the MTase active site, which inhibits both G-N-7 and 2’-O methylation, produces large polyadenylate in the presence or absence of SAH . This data confirms the earlier work demonstrating that the inhibition of cap methylation results in large polyadenylate. In contrast, several other substitutions that inhibit cap methylation, including D1762G, D1762N, G1672P, and G1675P, did not produce hyperpolyadenylated mRNA . Perhaps, K 1651A and D1762E substitutions might favor the binding of SAH at the SAM binding site in CR VI, resulting in hyperpolyadenylation without the need for supplemental SAH. Clearly, further studies are needed to understand the relationship between 5’ mRNA cap methyaltion and 3’ polyadenylation.
However, it appears clear that 5’ cap addition is required for 3’ polyadenylation, as evidenced by the polymerase mutants (G1154, T1157, H1227, and R1228) within CR V of L that inhibited cap addition also inhibit polyadenylation . These cap-defective polymerases synthesized truncated transcripts that predominantly terminated within the first 500 nt of the N gene and contained short A-rich sequences at their 3′ termini. To examine how the cap-defective polymerases respond to an authentic VSV termination and re-initiation signal present at each gene junction, a 382 nt gene was inserted at the leader-
7. Impact of mRNA cap methylation on viral replication and gene expression
In eukaryotic cells, it is well established that G-N-7 methylation of the mRNA cap structure is essential for mRNA stability and efficient translation [11, 48, 66]. Specifically, G-N-7 methylation of the mRNA cap structure is required for recognition of the cap by the rate limiting factor for translation initiation, eIF-4E [100, 101]. The mRNA cap structures of NNS RNA viruses are typically G-N-7 and 2’-O methylated. Although the precise mechanism by which VSV mRNAs are translated is unclear, they are broadly thought to utilize a variation of the canonical cap-dependent translational pathway [102-104]. In VSV-infected cells, host mRNA translation is rapidly inhibited through the suppression of the intracellular pools of eIF-4E by a manipulation of the phosphorylation status of the 4E binding protein (4E-BP1) . Nevertheless,
Based on the status of mRNA methylation, MTase-defective VSVs can be classified into three groups [14, 17, 84]. Viruses in the first group are completely defective in both G-N-7 and 2’-O methylation, including mutations in MTase active site (rVSV-K1651A, D1762A, K1795A, E1833Q, and E1833A), SAM binding site (rVSV-D1671V, G1675A, G4A, and G4AD), and putative RNA binding site (rVSV-Y1650A, F1691A, and E1764A). Viruses in the second group are specifically defective in G-N-7, but not 2’-O MTase, including mutants in SAM binding site (rVSV-G1670A, G1672A, and S1673A). Viruses in third group that require elevated SAM concentrations to permit full methylation including a mutant in SAM binding site (rVSV-G1674A) and putative RNA binding site (rVSV-Y1835A). With the exception of rVSV-G1674A and Y1835A, all MTase-defective VSVs were attenuated in cell culture as judged by diminished viral plaque size, reduced infectious viral progeny release (in single-step growth curves), and decreased levels of viral genomic RNA, mRNA, and protein synthesis. It appears that the degree of attenuation is consistent with the defects of the methylation. For example, viruses defective in both G-N-7 and 2’-O methylation had 2-5 log reductions in growth whereas viruses only defective in G-N-7 had 1-2 log declines in replication . Recombinant rVSV-G1674A and Y1835A replicated as efficiently as wild type rVSV . A remarkable finding is that some of the mutants in the SAM binding site (rVSV-G1675A, G4A, and G4AD) affected transcription and replication differently . For these mutants, replication was enhanced 2.5- to 4-fold, and transcription decreased up to 8-fold compared with rVSV. One feature of the gene expression strategy of NNS RNA viruses is that the polymerase complex controls two distinct RNA synthetic events: genomic RNA replication and mRNA transcription [20, 105, 106]. It is possible that SAM binding influences the switch of polymerase between replicase and transcriptase. Perhaps, L protein with SAM binding favors to function as transcriptase, whereas L protein that lacks SAM binding favors replicase function.
8. Impact of mRNA cap methylation on viral pathogenesis
Although it is well studied that MTase-defective viruses were attenuated in cell culture, the impact of mRNA cap methylation on viral pathogenesis
9. 2’-O methylation and innate immunity
While it is firmly established that G-N-7 methylation is essential for mRNA stability as well as efficient translation, the role(s) of ribose 2’-O methylation have proven more elusive. Recent studies on West Nile virus (WNV) suggest that the 2′-O methylation of the 5′ cap of viral RNA functions to evade innate host antiviral responses through escape of the suppression of interferon-stimulated genes, tetratricopeptide repeats (IFIT). Specifically, mutant WNV (E218A) defective in 2′-O MTase activity was attenuated in wild-type C57BL/6 mice, but remained pathogenic in knockout mice that lacked the type I interferon (IFN) signaling pathway. In addition, a vaccinia virus mutant (J3-K175R) and mouse hepatitis virus (MHV) mutant D130A, both of which lacked 2′-O MTase activity, exhibited enhanced sensitivities to the antiviral actions of IFN mediated by IFIT proteins. Interestingly, it was also reported that 2’-O methylation of mouse and human coronavirus RNA facilitates evasion from detection by the cytoplasmic RNA sensor Mda5 . Taken together, these studies suggest that 2’-O methylation of viral RNA provides a molecular signature for the discrimination of self and non-self mRNA. It is known that mRNAs of most NNS RNA viruses contain G-N-7 and 2’-O methylation. However, whether 2’-O methylation plays a similar role in all NNS RNA viruses is not known.
VSV is an excellent model to aid in the understanding the role of viral mRNA cap methylation in innate immunity. The mechanism of VSV mRNA cap methylation is unique in that 2’-O methylation precedes and facilitates the G-N-7 methylation [17, 18]. It is unknown why the order of VSV mRNA methylation is reversed compared to all known mRNA cap methylation reactions. One possibility is that the methylation of 2’-O allows VSV to successfully mimic cellular mRNA to avoid the detection by host innate immunity, which in turn promotes efficient viral replication in hosts. In fact, VSV is one of only a few viruses that replicates efficiently in a wide range of cell lines including mammalian cells, insect cells, and worms . Unlike WNV, VSV that is specifically defective in G-N-7 methylation can also be successfully recovered . VSV mutants defective in G-N-7 methylation or both G-N-7 and 2’-O methylations can serve as prototypes or controls to elucidate the role of methylation in innate immunity. In addition, the VSV mutant (rVSV-G4A) that was attenuated in cell culture retained low to moderate virulence, suggesting the possible role of methylation in averting the innate immune response . Notably, mRNAs of NDV, an avian paramyxovirus, are not 2’-O methylated . A direct comparison to determine whether 2’-O methylation of VSV has a similar biological function compared to WNV and MHV should prove very compelling.
10. MTase-defective viruses as live vaccine candidates
Recombinant viruses defective in MTase can be recovered from cloned full-length viral cDNA by a reverse genetics system. Viruses lacking MTase would likely be attenuated without affecting immunogenicity, since the MTase is located in L protein, which is not a neutralizing antibody target. Our group and others have identified a panel of MTase-defective VSV mutants which are attenuated in cell culture as well as in animal models [14, 17, 84, 107]. In addition, MTase-defective Sendai viruses also showed significant defects in viral growth in cell culture [91, 92]. By combining multiple substitutions within the methylation region, it should be possible to generate an attenuated virus that is genetically stable, as reversion to wild type at any single amino acid should not provide a fitness gain. Thus, ablating viral mRNA cap methylation would provide a new avenue to rationally attenuate these viruses for the development of live attenuated vaccines and exploit their use as viral vectors for vaccines, oncolytic therapy, and gene delivery. Recently, Ma et al., (2012) showed that MTase-defective VSVs were able to induce high levels of VSV-specific antibodies in mice and thus provided full protection against a virulent challenge with the VSV Indiana serotype . Recombinants rVSV-K1651A, D1762A, and E1833Q which were defective in both G-N-7 and 2’-O methylation, are attractive vaccine candidates since they are not only highly attenuated but also retain high immunogenicity. Although recombinants rVSV-G1670A and G1672A retained low virulence to mice, their pathogenicity was significantly reduced compared to rVSV. The safety of using these two viruses as live vaccine candidates necessitates further investigation.
Our studies on MTase-defective VSVs also shed light on developing live vaccine candidates for other NNS RNA viruses, particularly paramyxoviruses. Within paramyxoviruses,
RSV, hMPV, and PIV3 account for the majority of respiratory diseases infants, children, and the elderly [1-3]. However, there is no vaccine available for these important viruses. Recent research found that live attenuated vaccines are the most promising vaccine candidates for paramyxoviruses [1-3]. However, it has been technically challenging to isolate a virus with low virulence while retaining high immunogenicity. Introducing mutations in the MTase may provide a novel approach to generate live attenuated viruses for these viruses. It was reported that recombinant Sendai virus carrying point mutations in the MTase catalytic site (rSeV-K1782A) and the SAM binding site (rSeV-E1805A) were attenuated in cell culture . It will be of interesting to determine whether these Sendai recombinants are attenuated
11. mRNA cap methylation as a target for anti-viral drug discovery
It appears that the entire mRNA capping and methylation machinery in NNS RNA viruses is different from that of their hosts. This difference, coupled with the fact that replication of NNS RNA viruses occurs in the cytoplasm, suggests that mRNA cap formation is an excellent target for anti-viral drug discovery. Inhibition of the viral mRNA cap formation would likely inhibit downstream events such as replication, gene expression, viral spread, and ultimately viral infection. Since the mRNA of all NNS RNA viruses contains a methylated cap structure, classes of broadly active anti-viral agents may be developed by targeting the viral cap formation. For human RSV, several compounds were shown to inhibit polymerase activity which resulted in the synthesis of short uncapped transcripts . RSV mutants resistant to these inhibitors were selected and sequenced. It was found that these resistant mutants contained substitutions in CR V of L, specifically at E1269D, I1381S, and L1421F, suggesting that the mechanism of the action of these compounds is the inhibition of viral mRNA cap addition. Interestingly, these compounds showed strong antiviral activity against RSV infection in cell culture as well as in a mouse model, demonstrating that mRNA cap addition is an attractive antiviral target. It is known that SAH can inhibit viral mRNA cap methylation. Therefore, many adenosine analogues such as 3-deazaeplanocin-A are potent antiviral agents which can significantly inhibit VSV replication in cell culture [114, 115]. The mechanism of the action of these adenosine analogues is through the interference with the host enzyme SAH hydrolase that catalyzes the hydrolysis of SAH to adenosine and L-homocysteine. This reaction is reversible, and the products of this reaction are inhibitory to SAH hydrolase. Obviously, compounds that directly inhibit viral mRNA cap methylation are potent antiviral drugs. For example, sinefungin (SIN), a natural
12. Concluding remarks
In recent years, significant progress has been made in understanding the unusual mechanism of mRNA cap addition and methylation employed by VSV. First, VSV mRNA addition utilizes a novel PRNTase that transfers RNA to the GDP acceptor. Second, VSV mRNA cap methylation is catalyzed by a dual MTase that sequentially methylates the position 2’-O followed by G-N-7. Third, PRNTase and dual MTase have been mapped to single amino acid level in CR V and CR VI in the L protein, respectively. Finally, 5’ mRNA cap addition and methylation and 3’ polyadenylation are mechanistically and functionally linked. Apparently, the entire mRNA cap formation in VSV evolves a mechanism distinct to hosts. Thus, mRNA cap modification is an ideal target for vaccine and antiviral drug development. However, there are many questions need to be addressed. It is not known how polymerase coordinates nucleotide polymerization, mRNA cap addition, cap methylation, and polyadenylation. Although the general mechanism of mRNA modifications is defined, the detailed step in each reaction is still poorly understood. The GTPase required for mRNA capping has not been mapped in L protein. Besides the HR motif, it is not known which step of mRNA capping was affected by other mutations (such as T1157A and G1154A) in CR V of L protein. During mRNA cap methylation the exact mechanism by which the dual MTase methylates the 2’-O and G-N-7 is not known. The crystal structure is not known for L protein or any portion of L protein. The concept of using mRNA cap formation as antiviral target has been experimentally demonstrated, however, high-throughput screening methods toward drug discovery have not been developed. Furthermore, there is urgent need to understand the mRNA cap modification in other NNS RNA virus although it is speculated that they may also be achieved in a similar mechanism.