Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhao Y
PROVIDER: S-EPMC4580200 | biostudies-literature | 2015 Oct
REPOSITORIES: biostudies-literature
Publications
Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication.
Zhao Yongqian Y Soh Tingjin Sherryl TS Chan Kitti Wing Ki KW Fung Sarah Suet Yin SS Swaminathan Kunchithapadam K Lim Siew Pheng SP Shi Pei-Yong PY Huber Thomas T Lescar Julien J Luo Dahai D Vasudevan Subhash G SG
Journal of virology 20150812 20
We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate ...[more]
Similar Datasets
Project description:Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.
| S-EPMC4361662 | biostudies-literature
Project description:The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp in vitro. For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.
| S-EPMC6255901 | biostudies-literature
Project description:Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5'-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.
| S-EPMC4760774 | biostudies-literature
Project description:Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.
| S-EPMC4191377 | biostudies-literature
Project description:NS5 methyltransferase (Mtase) has a crucial role in the replication of dengue virus. There are two active sites on NS5 Mtase i.e., SAM and RNA-cap binding sites. Inhibition of the NS5 Mtase activity is expected to prevent the propagation of dengue virus. This study was conducted to design cyclic peptide ligands as enzyme inhibitors of dengue virus NS5 Mtase through computational approach. Cyclopentapeptides were designed as ligand of SAM binding site as much as 1635 and 736 cyclopentpeptides were designed as ligand of RNA-cap binding site. Interaction between ligand and NS5 Mtase has been conducted on the Docking simulation. The result shows that cyclopentapeptide CTWYC was the best peptide candidate on SAM binding site, with estimated free binding energy -30.72 kca/mol. Cyclopentapeptide CYEFC was the best peptide on RNA-cap binding site with estimated free binding energy -22.89 kcal/mol. Both peptides did not have tendency toward toxicity properties. So it is expected that both CTWYC and CYEFC ligands could be used as a potential antiviral drug candidates, which can inhibit the SAM and RNA-cap binding sites of dengue virus NS5 Mtase.
| S-EPMC3346019 | biostudies-literature
Project description:The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5' untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA70 and SLA80, which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides.IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5' untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle.
| S-EPMC5432874 | biostudies-literature
Project description:The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment.Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes.
| S-EPMC4886796 | biostudies-literature
Project description:Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.
| S-EPMC4976923 | biostudies-literature
Project description:Dengue is a viral infection caused by RNA infection of the family Flaviviridae and spread by the Aedes mosquitoes. Dengue NS5 methyltransferase is a known drug target for the disease. Therefore, it is of interest to design potential inhibitors for the target using molecular docking analysis. Our analysis shows the binding of compounds STOCK1N-98943, STOCK1N-98872, STOCK1N-98956, STOCK1N-98865, and STOCK1N-98950 with the protein drug target with optimal binding features for further in vitro and in vivo evaluations.
| S-EPMC6614123 | biostudies-literature
Project description:Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world's population, especially in the tropics and subtropics, is at risk of infection. Every year, 50-100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 μM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.
| S-EPMC6886872 | biostudies-literature