Viral RNA Cap Research Articles

Dengue Virus Non-Structural Protein 5.

The World Health Organization estimates that the yearly number of dengue cases averages 390 million. This mosquito-borne virus disease is endemic in over 100 countries and will probably continue spreading, given the observed trend in global warming. So far, there is no antiviral drug available against dengue, but a vaccine has been recently marketed. Dengue virus also serves as a prototype for the study of other pathogenic flaviviruses that are emerging, like West Nile virus and Zika virus. Upon viral entry into the host cell and fusion of the viral lipid membrane with the endosomal membrane, the viral RNA is released and expressed as a polyprotein, that is then matured into three structural and seven non-structural (NS) proteins. The envelope, membrane and capsid proteins form the viral particle while NS1-NS2A-NS2B-NS3-NS4A-NS4B and NS5 assemble inside a cellular replication complex, which is embedded in endoplasmic reticulum (ER)-derived vesicles. In addition to their roles in RNA replication within the infected cell, NS proteins help the virus escape the host innate immunity and reshape the host-cell inner structure. This review focuses on recent progress in characterizing the structure and functions of NS5, a protein responsible for the replication and capping of viral RNA that represents a promising drug target.

Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10.

The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that is thought to methylate the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap structures. This 2'-O-MTase activity is regulated by nsp10. The 2'-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2'-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7mGpppG (cap-0) into 7mGpppG2'Om (cap-1) RNA by 2'-OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2'-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2'-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2'-O methylation and to restore the host antiviral response. IMPORTANCE MERS-CoV codes for a cap 2'-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2'O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2'-O-methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2'-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.

Inhibition of Japanese encephalitis virus (JEV) replication by specific RNA aptamer against JEV methyltransferase

Japanese encephalitis virus (JEV) is the most common etiological agent of epidemic viral encephalitis. JEV encodes a single methyltransferase (MTase) domain located at the N-terminal region of the viral nonstructural protein NS5. JEV MTase is essential for viral replication and specifically catalyzes methylation of the viral RNA cap, which occurs exclusively in the cytoplasm. Therefore, JEV MTase is a potential target for antiviral therapy. Here, we identified specific and avid RNA aptamer (Kd ∼ 12 nM) with modified 2′-O-methyl pyrimidines against JEV MTase. The RNA aptamer efficiently inhibited viral cap methylation activity of MTase and interfered with JEV production in cells. Moreover, we generated a 24-mer truncated aptamer that could specifically bind to JEV MTase with high affinity (Kd ∼16 nM). The 24-mer aptamer efficiently inhibited JEV production and replication in cells. Therefore, MTase-specific RNA aptamer might be useful as an anti-JEV agent.

Structural Insight into NS5 of Zika Virus Leading to the Discovery of MTase Inhibitors.

Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28 341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 μM respectively, identifying lead compounds for anti-ZIKV drug development.

Inhibitor designing, virtual screening, and docking studies for methyltransferase: A potential target against dengue virus.

Aim:Aim of this work was to design and identify some S-adenosyl-L-homocysteine (SAH) analogs as inhibitors of S-adenosyl-L-methionine-dependent methyltransferase (MTase) protein using computational approaches.Introduction:According to the current scenario the dengue has been a global burden. The people are being killed by dengue virus in an abundant number. Despite of lot of research being going on dengue worldwide, there is no single drug which can kill its virus. This creates an urge for new drug target identification and designing. MTase has been reported as an effective target against dengue virus as it catalyzes an essential step in methylation and capping of viral RNA for viral replication.Materials and Methods:The crystal structure of MTase in complex with SAH was used for designing new analogs of SAH. SAH analogs designed were analyzed on the basis of docking, ADMET, and toxicity analysis done using Discovery Studio 3.5.Results:Seventeen analogs found noncarcinogenic, nonmutagenic, as well as good ADMET properties and good drug-like profile.Conclusion:These SAH analogs, inhibitors of MTase may act as drugs against dengue virus. Further synthesis and biological testing against dengue virus is under observation.

Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase.

The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2’-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition.

Electron microscopic analysis of rotavirus assembly-replication intermediates

Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30–70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly–replicase process.

Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase.

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5′ end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a Kd = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50 for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5′-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z′ of 0.83 and was successfully used to screen a library of known bioactives.

Computational analysis of methyl transfer reactions in dengue virus methyltransferase.

S-Adenosyl-L-methionine (SAM) dependent methyltransferases (MTases) play crucial roles in many biological processes. The MTase of the dengue virus is of particular interest for the development of antiviral drugs against flaviviruses. It catalyzes two distinct methylation reactions at the N7 and the 2'O position of the viral RNA cap structure. Based on density functional theory (DFT) electronic structure calculations, the molecular basis of the underlying chemical reactions involved in the N7 and the 2'O methyl transfer reactions of this enzyme were investigated using model systems. Calculations in the condensed phase show that both reactions are exergonic with significant activation barriers of 13.7 and 17.6 kcal/mol and stable product states, stabilized by 23.5 and 16.9 kcal/mol compared to the reactant states for the N7 and the 2'O reaction, respectively. We find that the reaction rate for the 2'O reaction is significantly enhanced in the presence of the native proton acceptor group, which lowers the activation barrier in the catalyzed reaction by 3.8 kcal/mol compared to the uncatalyzed reaction in aqueous solution. Furthermore, the 2'O reaction involves a methyl and a proton transfer reaction. Our results suggest that these two reactions occur in a concerted fashion in which the methyl group and the proton are transferred simultaneously. From a therapeutic viewpoint, SAM analogues stable under physiological conditions are particularly relevant. One such compound in MeAzaSAM, an isostructural mimic of SAM, for which the present calculations suggest that the methyl transfer reaction is unlikely to occur under biologically relevant conditions.

Flavivirus RNA methylation

The 5' end of eukaryotic mRNA contains the type-1 (m7GpppNm) or type-2 (m7GpppNmNm) cap structure. Many viruses have evolved various mechanisms to develop their own capping enzymes (e.g. flavivirus and coronavirus) or to 'steal' caps from host mRNAs (e.g. influenza virus). Other viruses have developed 'cap-mimicking' mechanisms by attaching a peptide to the 5' end of viral RNA (e.g. picornavirus and calicivirus) or by having a complex 5' RNA structure (internal ribosome entry site) for translation initiation (e.g. picornavirus, pestivirus and hepacivirus). Here we review the diverse viral RNA capping mechanisms. Using flavivirus as a model, we summarize how a single methyltransferase catalyses two distinct N-7 and 2'-O methylations of viral RNA cap in a sequential manner. For antiviral development, a structural feature unique to the flavivirus methyltransferase was successfully used to design selective inhibitors that block viral methyltransferase without affecting host methyltransferases. Functionally, capping is essential for prevention of triphosphate-triggered innate immune activation; N-7 methylation is critical for enhancement of viral translation; and 2'-O methylation is important for subversion of innate immune response during viral infection. Flaviviruses defective in 2'-O methyltransferase are replicative, but their viral RNAs lack 2'-O methylation and are recognized and eliminated by the host immune response. Such mutant viruses could be rationally designed as live attenuated vaccines. This concept has recently been proved with Japanese encephalitis virus and dengue virus. The findings obtained with flavivirus should be applicable to other RNA viruses.

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs

The flavivirus methyltransferase (MTase) sequentially methylates the N-7 and 2′-O positions of the viral RNA cap (GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA), using S-adenosyl-l-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC50 values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture.

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8. Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

2′-O Methylation of the Viral mRNA Cap by West Nile Virus Evades Ifit1-Dependent and -Independent Mechanisms of Host Restriction In Vivo

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 −/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD50 values in Ifit1 −/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 −/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8+ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.

Analysis of RNA Binding by the Dengue Virus NS5 RNA Capping Enzyme

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the KD for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5′ di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors: Implications for Antiviral Drug Development

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5' end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE-guanosine triphosphate (GTP) and CE-RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE-GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE-GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.