N-terminal Methyltransferase Research Articles

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins

Key messageAnalysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins.Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins.Graphical abstract

Characterizations of Protein Arginine Deiminase 1 as a Substrate of NTMT1: Implications of Nα-Methylation in Protein Stability and Interaction.

α-N-Methylation (Nα-methylation), catalyzed by protein N-terminal methyltransferases (NTMTs), constitutes a crucial post-translational modification involving the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the Nα-terminal amino group of substrate proteins. NTMT1/2 are known to methylate canonical Nα sequences, such as X-P-K/R. With over 300 potential human protein substrates, only a small fraction has been validated, and even less is known about the functions of Nα-methylation. This study delves into the characterizations of protein arginine deiminase 1 (PAD1) as a substrate of NTMT1. By employing biochemical and cellular assays, we demonstrated NTMT1-mediated Nα-methylation of PAD1, leading to an increase in protein half-life and the modulation of protein-protein interactions in HEK293T cells. The methylation of PAD1 appears nonessential to its enzymatic activity or cellular localization. Proteomic studies revealed differential protein interactions between unmethylated and Nα-methylated PAD1, suggesting a regulatory role for Nα-methylation in modulating PAD1's protein-protein interactions. These findings shed light on the intricate molecular mechanisms governing PAD1 function and expand our knowledge of Nα-methylation in regulating protein function.

Biological Relevance of Dual Lysine and N-Terminal Methyltransferase METTL13.

The dual methyltransferase methyltransferase-like protein 13, also referred to as METTL13, or formerly known as FEAT (faintly expressed in healthy tissues, aberrantly overexpressed in tumors), has garnered attention as a significant enzyme in various cancer types, as evidenced by prior literature reviews. Recent studies have shed light on new potential roles for METTL13, hinting at its promise as a therapeutic target. This review aims to delve into the multifaceted biology of METTL13, elucidating its proposed mechanisms of action, regulatory pathways, and its implications in disease states, as supported by the current body of literature. Furthermore, the review will highlight emerging trends and gaps in our understanding of METTL13, paving the way for future research efforts. By contextualizing METTL13 within the broader landscape of cancer biology and therapeutics, this study serves as an introductory guide to METTL13, aiming to provide readers with a thorough understanding of its role in disease phenotypes.

Docking and MM study of non-structural protein (NS5) of Japanese Encephalitis Virus (JEV) with some derivatives of adenosyl

Introduction: The flavivirus NS5, a non-structural protein of Japanese Encephalitis Virus (JEV), a serious deadly human pathogen responsible for epidemics in South East Asia, consists of N-terminal methyl transferase (MTase) domain and RNA-dependent RNA polymerase (RdRp) is known for unique viral genome replication and cap formation activity. S-adenosyl executes a crucial function in these viral activities. S-adenosyl derivatives are chosen as potential binders with the MTase domain of NS5 based on MM and docking studies.Methods: MM GBSA (Generalized Born Surface Area) simulation were performed to evaluate the binding energy, following the 100 nanosecond (ns) production MD simulation in the periodic boundary condition (PBC) for the selected docked ligands with NS5. Quasi-harmonic entropy of the ligands was also calculated with semi-empirical calculations at the PM3/PM6 level supporting docking and MM-GBSA results.Results and discussion: The residue-wise decomposition energy reveals that the key hydrophobic residues Gly 81, Phe 133, and Ile 147 in the RdRp-MTase interface, indicate the biological relevance. These residues act as the key residue stabilizer, binding vigorously with S-Adenosyl derivatives in the vicinity of the interface between the MTase domain and RdRp. This paves the way for the other potential drug as an inhibitor for the enzymatic activity of the NS5.

The comprehensive analysis of the prognostic and functional role of N-terminal methyltransferases 1 in pan-cancer.

NTMT1, a transfer methylase that adds methyl groups to the N-terminus of proteins, has been identified as a critical player in tumor development and progression. However, its precise function in pan-cancer is still unclear. To gain a more comprehensive understanding of its role in cancer, we performed a thorough bioinformatics analysis. To conduct our analysis, we gathered data from multiple sources, including RNA sequencing and clinical data from the TCGA database, protein expression data from the UALCAN and HPA databases, and single-cell expression data from the CancerSEA database. Additionally, we utilized TISIDB to investigate the interaction between the tumor and the immune system. To assess the impact of NTMT1 on the proliferation of SNU1076 cells, we performed a CCK8 assay. We also employed cellular immunofluorescence to detect DNA damage and used flow cytometry to measure tumor cell apoptosis. Our analysis revealed that NTMT1 was significantly overexpressed in various types of tumors and that high levels of NTMT1 were associated with poor survival outcomes. Functional enrichment analysis indicated that NTMT1 may contribute to tumor development and progression by regulating pathways involved in cell proliferation and immune response. In addition, we found that knockdown of NTMT1 expression led to reduced cell proliferation, increased DNA damage, and enhanced apoptosis in HNSCC cells. High expression of NTMT1 in tumors is associated with poor prognosis. The underlying regulatory mechanism of NTMT1 in cancer is complex, and it may be involved in both the promotion of tumor development and the inhibition of the tumor immune microenvironment.

The SET and ankyrin domains of the secreted Legionella pneumophila histone methyltransferase work together to modify host chromatin.

Legionella pneumophila is an intracellular bacterium responsible of Legionnaires' disease, a severe pneumonia that is often fatal when not treated promptly. The pathogen's ability to efficiently colonize the host resides in its ability to replicate intracellularly. Essential for intracellular replication is translocation of many different protein effectors via a specialized secretion system. One of them, called RomA, binds and directly modifies the host chromatin at a unique site (tri-methylation of lysine 14 of histone H3 [H3K14me]). However, the molecular mechanisms of binding are not known. Here, we resolve this question through structural characterization of RomA together with the H3 peptide. We specifically reveal an active role of the ankyrin repeats located in its C-terminal in the interaction with the histone H3 tail. Indeed, without the ankyrin domains, RomA loses its ability to act as histone methyltransferase. These results discover the molecular mechanisms by which a bacterial histone methyltransferase that is conserved in L. pneumophila strains acts to modify chromatin.

More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Inter-domain coordination essential for dengue virus non-structural (5) NS5 interaction with stem loop a (SLA).

Dengue virus is the most prevalent arthropod-borne virus and is a part of the flavivirus family. There are four known dengue serotypes, and humans can be infected with more than one serotype which can lead to severe dengue. There are no clinically approved antiviral drugs for the treatment of dengue infection to date, and vaccines available are limited to seropositive individuals. The non-structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is a major component of the viral replication complex. Dengue NS5 has an N-terminal methyltransferase (MTase) domain responsible for 5’ RNA capping, and a C-terminal RNA-dependent-RNA-polymerase (RdRp) domain responsible for de novo RNA synthesis. Dengue NS5 is also known to interact with RNA elements in the 5’-untranslated regions to promote viral RNA synthesis, in particular the 5’ stem loop A (SLA). However, there is limited structural characterization of the dengue NS5/SLA complex. Here, we characterized the interaction of the full length and individual domains of dengue serotype 2 (DENV2) NS5 with SLA at the 5’-UTR using surface plasmon resonance (SPR) studies, electromobility shift assays (EMSAs) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). HDX-MS studies were used to probe the structural dynamics of DENV2 NS5 and its binding interface with SLA. Results from these studies suggest that coordination is required between the MTase and RdRp domains, which stabilize the interaction between NS5 and SLA. Ultimately, our results will shed more light into how dengue NS5 interacts with SLA to discriminate viral RNA from cellular RNA.

Three's a crowd - why did three N-terminal methyltransferases evolve for one job?

N-terminal methylation of the α-amine group (Nα-methylation) is a post-translational modification (PTM) that was discovered over 40 years ago. Although it is not the most abundant of the Nα-PTMs, there are more than 300 predicted substrates of the three known mammalian Nα-methyltransferases, METTL11A and METTL11B (also known as NTMT1 and NTMT2, respectively) and METTL13. Of these ∼300 targets, the bulk are acted upon by METTL11A. Only one substrate is known to be Nα-methylated by METTL13, and METTL11B has no proven in vivo targets or predicted targets that are not also methylated by METTL11A. Given that METTL11A could clearly handle the entire substrate burden of Nα-methylation, it is unclear why three distinct Nα-methyltransferases have evolved. However, recent evidence suggests that many methyltransferases perform important biological functions outside of their catalytic activity, and the Nα-methyltransferases might be part of this emerging group. Here, we describe the distinct expression, localization and physiological roles of each Nα-methyltransferase, and compare these characteristics to other methyltransferases with non-catalytic functions, as well as to methyltransferases with both catalytic and non-catalytic functions, to give a better understanding of the global roles of these proteins. Based on these comparisons, we hypothesize that these three enzymes do not just have one common function but are actually performing three unique jobs in the cell.

Structure-Activity Relationship Studies of Venglustat on NTMT1 Inhibition.

The protein N-terminal methyltransferase 1 (NTMT1) is implicated in neurogenesis, retinoblastoma, and cervical cancer. However, its pharmacological potentials have not been elucidated due to the lack of drug-like inhibitors. Here, we report the discovery of the first NTMT1 in vivo chemical probe GD433 by structure-guided optimization of our previously reported lead compound venglustat. GD433 (IC50 = 27 ± 1.1 nM) displays improved potency and selectivity than venglustat across biochemical, biophysical, and cellular assays. GD433 also displays good oral bioavailability and can serve as an in vivo chemical probe to dissect the pharmacological roles of Nα methylation. In addition, we also identified a close analogue (YD2160) that is inactive against NTMT1. The active inhibitor and negative control will serve as valuable tools to examine the physiological and pharmacological functions of NTMT1 catalytic activity.

Optimizing purification and activity assays of N-terminal methyltransferase complexes.

In vitro methyltransferase assays have traditionally been carried out with tritiated S-adenosyl-methionine (SAM) as the methyl donor, as site-specific methylation antibodies are not always available for Western or dot blots and structural requirements of many methyltransferases prohibit the use of peptide substrates in luminescent or colorimetric assays. The discovery of the first N-terminal methyltransferase, METTL11A, has allowed for a second look at non-radioactive in vitro methyltransferase assays, as N-terminal methylation is amenable to antibody production and the limited structural requirements of METTL11A allow for its methylation of peptide substrates. We have used a combination of Western blots and luminescent assays to verify substrates of METTL11A and the two other known N-terminal methyltransferases, METTL11B and METTL13. We have also developed these assays for use beyond substrate identification, showing that METTL11A activity is opposingly regulated by METTL11B and METTL13. Here we provide two methods for non-radioactive characterization of N-terminal methylation, Western blots with full-length recombinant protein substrates and luminescent assays with peptide substrates, and describe how each can be additionally adapted to look at regulatory complexes. We will review the advantages and disadvantages of each method in context with the other types of in vitro methyltransferase assays and discuss why these types of assays could be of general use to the N-terminal modification field.

Site-specific methylation on α-N-terminus of peptides through chemical and enzymatic methods.

Protein α-N-terminal (Nα) methylation is a post-translational modification catalyzed by N-terminal methyltransferase 1/2 (NTMT1/2) and METTL13. Nα methylation affects protein stability, protein-protein interaction, and protein-DNA interaction. Thus, Nα methylated peptides are essential tools to study the function of Nα methylation, generate specific antibodies for different states of Nα methylation, and characterize the enzyme kinetics and activity. Here, we describe chemical methods of site-specific synthesis of Nα mono-, di-, and trimethylated peptides in the solid phase. In addition, we describethe preparation of trimethylation peptides by recombinant NTMT1 catalysis.

Sofosbuvir and its tri-phosphate metabolite inhibit the RNA-dependent RNA polymerase activity of non-structural protein 5 from the Kyasanur forest disease virus

Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.

Design and characterization of PROTAC degraders specific to protein N-terminal methyltransferase 1

Protein N-terminal methylation catalyzed by N-terminal methyltransferase 1 (NTMT1) is an emerging methylation present in eukaryotes, playing important regulatory roles in various biological and cellular processes. Although dysregulation of NTMT1 has been linked to many diseases such as colorectal cancer, their molecular and cellular mechanisms remain elusive due to inaccessibility to an effective cellular probe. Here we report the design, synthesis, and characterization of the first-in-class NTMT1 degraders based on proteolysis-targeting chimera (PROTAC) strategy. Through a brief structure-activity relationship (SAR) study of linker length, a cell permeable degrader 1 involving a von Hippel-Lindau (VHL) E3 ligase ligand was developed and demonstrated to reduce NTMT1 protein levels effectively and selectively in time- and dose-dependent manners in colorectal carcinoma cell lines HCT116 and HT29. Degrader 1 displayed DC50 = 7.53 μM and Dmax > 90% in HCT116 (cellular IC50 > 100 μM for its parent inhibitor DC541). While degrader 1 had marginal cytotoxicity, it displayed anti-proliferative activity in 2D and 3D culture environment, resulting from cell cycle arrested at G0/G1 phase in HCT116. Label-free global proteomic analysis revealed that degrader 1 induced overexpression of calreticulin (CALR), an immunogenic cell death (ICD) signal protein that is known to elicit antitumor immune response and clinically linked to a high survival rate of patients with colorectal cancer upon its upregulation. Collectively, degrader 1 offers the first selective cellular probe for NTMT1 exploration and a new drug discovery modality for NTMT1-related oncology and diseases.

Venglustat Inhibits Protein N-Terminal Methyltransferase 1 in a Substrate-Competitive Manner.

Venglustat is a known allosteric inhibitor for ceramide glycosyltransferase, investigated in diseases caused by lysosomal dysfunction. Here, we identified venglustat as a potent inhibitor (IC50 = 0.42 μM) of protein N-terminal methyltransferase 1 (NTMT1) by screening 58,130 compounds. Furthermore, venglustat exhibited selectivity for NTMT1 over 36 other methyltransferases. The crystal structure of NTMT1-venglustat and inhibition mechanism revealed that venglustat competitively binds at the peptide substrate site. Meanwhile, venglustat potently inhibited protein N-terminal methylation levels in cells (IC50 = 0.5 μM). Preliminary structure-activity relationships indicated that the quinuclidine and fluorophenyl parts of venglustat are important for NTMT1 inhibition. In summary, we confirmed that venglustat is a bona fide NTMT1 inhibitor, which would advance the study on the biological roles of NTMT1. Additionally, this is the first disclosure of NTMT1 as a new molecular target of venglustat, which would cast light on its mechanism of action to guide the clinical investigations.

Role of the homologous MTase-RdRp interface of flavivirus intramolecular NS5 on duck tembusu virus

Flavivirus nonstructural protein 5 (NS5) harbors the N-terminal methyltransferase (MTase) and C-terminal polymerase RNA-dependent RNA polymerase (RdRp). The intramolecular NS5 features an integral MTase and RdRp interface with two components: a six-residue hydrophobic network and a GTR linker. Herein, the determinants of the MTase-RdRp interface and flavivirus substituted GTR linker were explored in TMUV replication and proliferation. First, the NanoLuc® Binary Technology (NanoBiT) and coimmunoprecipitation assays (Co-IP) methods confirmed the interaction between the MTase and RdRp domains of TMUV NS5. To screen for an optimal orientation for reporter gene fusion to the protein of interest, the signal activity of eight combinations of MTase and RdRp was explored. Intriguingly, all the combinations with the reporter gene fused to the C-terminal of MTase (1.1 C/2.1 C MTase) could barely detect any positive signal, suggesting a role for the GTR linker of the MTase C-terminal in MTase-RdRp affinity. Based on the flavivirus NS5 homologous interplay, we introduced alanine mutations into the MTase-RdRp interface of TMUV NS5. However, no single or pairwise mutation was found to abort the NS5 intramolecular interaction. Then, a mutated replicon and infectious clone were constructed to analyze the replication ability and properties of the recombinant virus. The mutant replicons of MTase F113A and M115A replicated to comparable extent as the wild type (WT). However, the replication level of the mutant MTase W121A was impaired without an obvious decrease in proliferation and virulence. Both the RdRp F351A and P585A mutants could replicate and proliferate well. Notably, the RdRp F467A virus was attenuated and did not strikingly impair the MTase-RdRp interaction. Furthermore, the TMUV was specifically compatible with the substituted NS5 with a Japanese encephalitis virus (JEV) GTR linker. Compensatory mutations were observed in the context of a defective MTase-RdRp interface after several passages of the rescued mutants in BHK-21 cells. A greater understanding of the molecular mechanism of the NS5 protein controlling duck TMUV replication will facilitate the design of novel therapies.

Improved Cell-Potent and Selective Peptidomimetic Inhibitors of Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferase 1 (NTMT1) recognizes a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) and transfers 1–3 methyl groups to the N-terminal region of its substrates. Guided by the co-crystal structures of NTMT1 in complex with the previously reported peptidomimetic inhibitor DC113, we designed and synthesized a series of new peptidomimetic inhibitors. Through a focused optimization of DC113, we discovered a new cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM). GD562 exhibited improved inhibition of the cellular N-terminal methylation levels of both the regulator of chromosome condensation 1 and the oncoprotein SET with an IC50 value of ~50 µM in human colorectal cancer HCT116 cells. Notably, the inhibitory activity of GD562 for the SET protein increased over 6-fold compared with the previously reported cell-potent inhibitor DC541. Furthermore, GD562 also exhibited over 100-fold selectivity for NTMT1 against several other methyltransferases. Thus, this study provides a valuable probe to investigate the biological functions of NTMT1.

Insights into the structural dynamics and RNA binding of the dengue nonstructural 5 (NS5) protein

Dengue virus is the most prevalent arthropod-borne virus. It is a part of the flavivirus family and infections with dengue virus can lead to severe dengue disease. There are no clinically approved antiviral drugs for the treatment of dengue infection to date, and vaccines available are limited to seropositive individuals. The non-structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is responsible for the replication of the viral RNA genome. NS5 has an N-terminal methyltransferase (MTase) domain responsible for 5’ RNA capping, and a C-terminal RNA-dependent-RNA-polymerase (RdRp) domain responsible for de novo RNA synthesis.