N-terminal Methyltransferase Research Articles

Identification of Possible Inhibitor Molecule against NS5 MTase and RdRp Protein of Dengue Virus in Saudi Arabia

Abstract: Background: Non-structural protein 5 (NS5) is considered as an important protein in Dengue viruses (DENVs). It contains N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. NS5 plays a crucial role in the replication of Dengue viruses. Therefore it is considered as an attractive candidate for the development of therapeutics in anti-viral infections and diseases. Aim: The aim of proposed in-silico study was to to screen and identify potential lead molecules with drug like properties to inhibit the activity of NS5 protein in Dengue virus infections. Materials and Methods: Computational bioinformatics analysis was implemented to identify the lead molecules with inhibition activity against MTase and RdRp domains of Dengue virus protein NS5. Phytochemicals and active bioassay compounds were screened based on their reported antiviral mactivities. A total of fifty-one natural compounds and one hundred active compounds were selected by evaluating eighty one bioassay studies. Results: Results of the current study revealed galactomannan, galactan, hyperside, carrageenan, tetrahydroxy, lamdacarragenon, zosteric acid, trihydroxy, quercetin and sulfoximine as top inhibitors of the Dengue virus NS5 protein. Lead molecules namely lamdacarragenon, carrageenan, balsacanea, galactan, trihydroxy, hyperside, myricetin, glycyrrhiza, isosilandrin, rhodiola and silyhermin showed the utmost hydrogen bonding. Overall, we observed that the bioassay active compounds showed less interaction with the MTase and RdRp domains of NS5 protein as compared to natural ligands. Conclusion: Based on the findings of current study, we concluded that the phytochemicals are the most favourable among the docked molecules that showed a significant inhibitory activity against the MTase and RdRp domains of NS5 protein of dengue viruses. We suggest that these lead molecules can be validated experimentally and implemented for the development of therapeutics in viral infections like Dengue. Key words: Dengue virus, Molecular docking, Molecular structure, MTase, NS5, Non-structural viral proteins, RdRp.

Age-related neurodegeneration and cognitive impairments of NRMT1 knockout mice are preceded by misregulation of RB and abnormal neural stem cell development

N-terminal methylation is an important posttranslational modification that regulates protein/DNA interactions and plays a role in many cellular processes, including DNA damage repair, mitosis, and transcriptional regulation. Our generation of a constitutive knockout mouse for the N-terminal methyltransferase NRMT1 demonstrated its loss results in severe developmental abnormalities and premature aging phenotypes. As premature aging is often accompanied by neurodegeneration, we more specifically examined how NRMT1 loss affects neural pathology and cognitive behaviors. Here we find that Nrmt1−/− mice exhibit postnatal enlargement of the lateral ventricles, age-dependent striatal and hippocampal neurodegeneration, memory impairments, and hyperactivity. These morphological and behavior abnormalities are preceded by alterations in neural stem cell (NSC) development. Early expansion and differentiation of the quiescent NSC pool in Nrmt1−/− mice is followed by its subsequent depletion and many of the resulting neurons remain in the cell cycle and ultimately undergo apoptosis. These cell cycle phenotypes are reminiscent to those seen with loss of the NRMT1 target retinoblastoma protein (RB). Accordingly, we find misregulation of RB phosphorylation and degradation in Nrmt1−/− mice, and significant de-repression of RB target genes involved in cell cycle. We also identify novel de-repression of Noxa, an RB target gene that promotes apoptosis. These data identify Nα-methylation as a novel regulatory modification of RB transcriptional repression during neurogenesis and indicate that NRMT1 and RB work together to promote NSC quiescence and prevent neuronal apoptosis.

Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 310 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations. Using reverse genetics system, we either introduced rigidity in the linker through mutation to a proline or flexibility through a glycine mutation at position 266. ZIKV NS5 Val 266 to Pro mutation was lethal for viral RNA replication while the Gly mutation was severely attenuated. Serial passaging of cell culture supernatant derived from C6/36 mosquito cells transfected with mutant ZIKV RNA showed that the attenuation can be rescued. Next generation deep sequencing revealed four single nucleotide polymorphisms that occur with an allele frequency >98%. The single non-synonymous NS5 mutation Glu419 to Lys is adjacent to RdRp motif G at the tip of the fingers subdomain, while the remaining three are synonymous variants at nucleotide positions 1403, 4403 and 6653 in the genome. Reverse engineering the changes into the ZIKV NS5/Val266Gly background followed by serial passaging revealed that residue 266 is under strong positive selection to revert back to Val. The interaction of the specific conformation of the NS5 linker with Val at position 266 and the RNA binding motif G region may present a potential strategy for allosteric antiviral drug development.

Structure, Activity and Function of the Dual Protein Lysine and Protein N-Terminal Methyltransferase METTL13.

METTL13 (also known as eEF1A-KNMT and FEAT) is a dual methyltransferase reported to target the N-terminus and Lys55 in the eukaryotic translation elongation factor 1 alpha (eEF1A). METTL13-mediated methylation of eEF1A has functional consequences related to translation dynamics and include altered rate of global protein synthesis and translation of specific codons. Aberrant regulation of METTL13 has been linked to several types of cancer but the precise mechanisms are not yet fully understood. In this article, the current literature related to the structure, activity, and function of METTL13 is systematically reviewed and put into context. The links between METTL13 and diseases, mainly different types of cancer, are also summarized. Finally, key challenges and opportunities for METTL13 research are pinpointed in a prospective outlook.

Chemoproteomic Study Uncovers HemK2/KMT9 As a New Target for NTMT1 Bisubstrate Inhibitors.

Understanding the selectivity of methyltransferase inhibitors is important to dissecting the functions of each methyltransferase target. From this perspective, we report a chemoproteomic study to profile the selectivity of a potent protein N-terminal methyltransferase 1 (NTMT1) bisubstrate inhibitor NAH-C3-GPKK (Ki,app = 7 ± 1 nM) in endogenous proteomes. First, we describe the rational design, synthesis, and biochemical characterization of a new chemical probe 6, a biotinylated analogue of NAH-C3-GPKK. Next, we systematically analyze protein networks that may selectively interact with the biotinylated probe 6 in concert with the competitor NAH-C3-GPKK. Besides NTMT1, the designated NTMT1 bisubstrate inhibitor NAH-C3-GPKK was found to also potently inhibit a methyltransferase complex HemK2-Trm112 (also known as KMT9-Trm112), highlighting the importance of systematic selectivity profiling. Furthermore, this is the first potent inhibitor for HemK2/KMT9 reported until now. Thus, our studies lay the foundation for future efforts to develop selective inhibitors for either methyltransferase.

CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation

ABSTRACT The N-terminal methyltransferase NRMT1 is an important regulator of protein/DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked Pax7 expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.

Deciphering the role of N‐terminal methylation in modulating yeast protein function including the multitasking stress response protein, Hsp31

As the one of the most common protein post-translational modifications (PTMs), protein methylation, especially a-N-methylation is largely underexplored. a-N-methylation is conserved in both eukaryotes and prokaryotes. Identification of the two a-N-methyltransferases in humans, NTMT1 and NTMT2, and their homologue of high sequence similarity in yeast, Tae1, indicated a shared novel protein regulation mechanism in both species. Recently, NTMT1/2 are shown important functions in many biological pathways including cell division and dysregulation is implicated in cancer development. Structural studies and in vitro analysis support a canonical motif, X1-P2-K3 (X=A, S, P or G after initiating M is cleaved) on substrate proteins as the determinant for recognition by NTMT1/2 and Tae1. However, a-N-methylation on most of the potential substrates with the motif are not substantiated. The object of this study is using yeast as an ideal model to investigate the a-N-terminal methylome, taking advantage of its well observed phenotypes, fully sequenced genome, tractable genetics, and comparatively simplified N-terminal methyltransferase network. We combine bioinformatic searching, mass spectrometry PTM mapping, yeast phenotypic assay and biochemical assays in this study to approach our aims: (1) Screen for potential substrates and identify methylation events using a proteomic dataset repurposing approach, (2) Confirm a-N-methylation experimentally, (3) Investigate the Tae1 relevant phenotypes and biological pathways. (4) Exploration of mechanism in regulating substrate function. Initial bioinformatic research by motif mapping indicates that 45 proteins might be regulated by Tae1 through a-N-methylation. We have verified a-N-methylation on 6 proteins including Heat shock protein 31, Hsp31. Hsp31 and its three paralogues are members of the DJ-1/ThiJ/PfpI family with the human protein implicated in Parkinson's Disease. We demonstrate that TAE1 deficient yeast show susceptibility to inhibitors of protein synthesis or microtubule assembly. However, the same strain demonstrates resistance to heat stress and oxidative stress. Hsp31 is a multifunctional protein with chaperone, deglycase and methylglyoxalase activity. Thus, we hypothesize that Tae1 regulates yeast response to heat stress and oxidative stress potentially through its substrate, Hsp31. Further studies confirmed that Tae1 is responsible for a-N-methylation of Hsp31. Overexpression of Hsp31 N-terminal mutants with decreased methylation potential show elevated protective effect under both heat shock and methylglyoxalase (MGO) stress. However, MGO stress treatment on yeast show limited effect on methylglyoxalase activity of purified Hsp31. The exact mechanism of how a-N-methylation affects protein function is currently under assessment for Hsp31 and other potential substrates. The results of this study will reveal the more global view of N-terminal methyltransferase substrates, facilitate the translation to human substrates and further assist in investigating N-terminal methylation under various disease contexts and assist in optimizing drug design of disease targets.

Structure-based Discovery of Cell-Potent Peptidomimetic Inhibitors for Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferases (NTMTs) catalyze the methylation of the α-N-terminal amines of proteins starting with an X-P-K/R motif. NTMT1 has been implicated in various cancers and in aging, implying its role as a potential therapeutic target. Through structural modifications of a lead NTMT1 inhibitor, BM30, we designed and synthesized a diverse set of inhibitors to probe the NTMT1 active site. The incorporation of a naphthyl group at the N-terminal region and an ortho-aminobenzoic amide at the C-terminal region of BM30 generates the top cell-potent inhibitor DC541, demonstrating increased activity on both purified NTMT1 (IC50 of 0.34 ± 0.02 μM) and the cellular α-N-terminal methylation level of regulator of chromosome condensation 1 (RCC1, IC50 value of 30 μM) in human colorectal cancer HT29 cells. Furthermore, DC541 exhibits over 300-fold selectivity to several methyltransferases. This study points out the direction for the development of more cell-potent inhibitors for NTMT1.

METTL16, Methyltransferase-Like Protein 16: Current Insights into Structure and Function.

Methyltransferase-like protein 16 (METTL16) is a human RNA methyltransferase that installs m6A marks on U6 small nuclear RNA (U6 snRNA) and S-adenosylmethionine (SAM) synthetase pre-mRNA. METTL16 also controls a significant portion of m6A epitranscriptome by regulating SAM homeostasis. Multiple molecular structures of the N-terminal methyltransferase domain of METTL16, including apo forms and complexes with S-adenosylhomocysteine (SAH) or RNA, provided the structural basis of METTL16 interaction with the coenzyme and substrates, as well as indicated autoinhibitory mechanism of the enzyme activity regulation. Very recent structural and functional studies of vertebrate-conserved regions (VCRs) indicated their crucial role in the interaction with U6 snRNA. METTL16 remains an object of intense studies, as it has been associated with numerous RNA classes, including mRNA, non-coding RNA, long non-coding RNA (lncRNA), and rRNA. Moreover, the interaction between METTL16 and oncogenic lncRNA MALAT1 indicates the existence of METTL16 features specifically recognizing RNA triple helices. Overall, the number of known human m6A methyltransferases has grown from one to five during the last five years. METTL16, CAPAM, and two rRNA methyltransferases, METTL5/TRMT112 and ZCCHC4, have joined the well-known METTL3/METTL14. This work summarizes current knowledge about METTL16 in the landscape of human m6A RNA methyltransferases.

Modulation of N-terminal methyltransferase 1 by an N6-methyladenosine-based epitranscriptomic mechanism

Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N6-methyladenosine (m6A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m6A. Together, these results revealed a novel m6A-based epitranscriptomic mechanism in regulating protein N-terminal methylation.

Selective Peptidomimetic Inhibitors of NTMT1/2: Rational Design, Synthesis, Characterization, and Crystallographic Studies.

Protein N-terminal methyltransferases (NTMTs) methylate the α-N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitor for NTMT1/2. Biochemical and cocrystallization studies manifest that BM30 (with a half-maximal inhibitory concentration of 0.89 ± 0.10 μM) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 MTs, indicating its potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analogue DC432 (IC50 of 54 ± 4 nM) decreases the N-terminal methylation level of the regulator of chromosome condensation 1 and SET proteins in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in the future.

Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogues.

The bisubstrate analogue strategy is a promising approach to develop potent and selective inhibitors for protein methyltransferases. Herein, the interactions of a series of bisubstrate analogues with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the active site of NTMT1. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analogue to occupy both substrate- and cofactor-binding sites of NTMT1, but the bisubstrate analogue with a 5-C atom linker only interacts with the substrate-binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki,app = 130 ± 40 pM) for NTMT1 to date, displaying more than 3000-fold selectivity for other methyltransferases and even for its homologue NTMT2. This study reveals the molecular basis for the plasticity of the active site of NTMT1. Additionally, our study outlines general guidance on the development of bisubstrate inhibitors for any methyltransferases.

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors.

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

Mechanistic insights into m6A modification of U6 snRNA by human METTL16.

The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5′-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16–U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.

Structure-based screening and validation of bioactive compounds as Zika virus methyltransferase (MTase) inhibitors through first-principle density functional theory, classical molecular simulation and QM/MM affinity estimation

Recent Zika virus (ZIKV) outbreak and association with human diseases such as neurological disorders have raised global health concerns. However, in the absence of an approved anti-ZIKV drug has generated urgency for the drug development against ZIKV infection. Here, structure-based virtual screening of 8589 bioactive compounds, screened at the substrate-binding site of ZIKV nonstructural 5 (NS5)-based structure N-terminal methyltransferase (MTase) domain followed by ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling concluded the four potential lead inhibitors, i.e. (4-acetylamino-benzenesulfonylamino)-acetic acid (F3342-0450), 3-(5-methylfuran-2-yl)-N-(4-sulfamoylphenyl)propanamide (F1736-0142), 8-(2-hydroxy-ethylamino)-1,3-dimethyl-7-(3-methyl-benzyl)-3,7-dihydro-purine-2,6-dione (F0886-0080) and N-[4-(aminosulfonyl)phenyl]-2,3-dihydro-1,4-benzodioxine-2-carboxamide (F0451-2187). Collectively, extra precision docking and Density Functional Theory(DFT) calculations studies identified the F3342-0450 molecule, having strong interactions on the active site of MTase, further supported by molecular dynamics simulation, binding affinity and hybrid QM/MM calculations, suggest a new drug molecule for the antiviral drug development against ZIKV infection. Communicated by Ramaswamy H. Sarma

Flavivirus RNA-Dependent RNA Polymerase Interacts with Genome UTRs and Viral Proteins to Facilitate Flavivirus RNA Replication.

Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses. Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

Advance in research on structure and functions of the nonstructural protein 5 of Zika virus

Zika virus (ZIKV) is an important member of genus Flavivirus which includes dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. ZIKV has attracted worldwide attention because of its unexpected causal link to microcephaly, Guillain-Barre syndrome, and other serious neurological diseases. So far, it has been spreading to 90 countries or territories worldwide, and approximately 3.6 billion people are now living at risk of ZIKV infection. Although a lot of efforts have been made to understand the pathogenesis of ZIKV, there is no approved vaccines or antiviral drugs available. ZIKV has a single-stranded and positive-sense genome RNA, which contains a single open reading frame flanked by the highly structured untranslated regions (UTRs). The ORF encodes a polyprotein precursor which is further cleaved into 3 structural proteins (C, prM/M and E) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by either host or viral protease. ZIKV NS5 is the largest and the most conserved protein among all viral proteins. ZIKV NS5 can be divided into two domains: the N-terminal methyltransferase (MTase) domain and C-terminal RNA-dependent RNA polymerase (RdRp) domain. Recently, the high-resolution structure of full-length ZIKV NS5 protein as well as the individual MTase and RdRp domains have been resolved, which led to better understanding of its biological function. As a multifunctional protein, ZIKV NS5 interacts with several cis -acting RNA elements located in the 5′UTR to initiate viral RNA synthesis, and adds the 5′RNA cap structure to facilitate translation of the polyprotein. NS5 is also involved in the regulation of host innate antiviral immune responses. As such, ZIKV NS5 is widely considered to be a potential target for antiviral drug design, and the research towards this goal also accelerated our understanding of its structure and function. A panel of small molecule inhibitors targeting ZIKV NS5 has been identified to have ideal antiviral effects in both cell cultures and animal models. This review summarizes recent advances on the structure and biological functions of ZIKV NS5 protein.

Expression and purification of the truncated duck DTMUV NS5 protein and the subcellular localization of NS5 in vitro

Duck Tembusu virus (DTMUV) non-structural protein 5 (NS5), which harbors an N-terminal methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain, is central to virus replication. In this study, a sequence encoding amino acid residues 1-408 of NS5 was amplified and ligated into a pET32a vector to generate a pET32a-NS51-408 expression vector. Recombinant protein was expressed, purified, and used to arise a mouse anti-NS5 specific polyclonal antibody (DTMUV-NS51-408 mPAb). Indirect immunofluorescence assays indicated that NS5 protein localized within the cytoplasmic region of DTMUV-infected or transfected cells; the localization was not affected in the presence of a nuclear export inhibitor. This study provides the first demonstration that, in contrast to the data reported for NS5 proteins of Dengue and Yellow fever virus, DTMUV NS5 had slight nuclear localization activity.

Supramolecular arrangement of the full-length Zika virus NS5.

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.