The Experts below are selected from a list of 13410 Experts worldwide ranked by ideXlab platform
Pei Yong Shi - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus Nonstructural Protein 5 (NS5)
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Yongqian Zhao, Subhash G. Vasudevan, Tingjin Sherryl Soh, Siew Pheng Lim, Ka Yan Chung, Kunchithapadam Swaminathan, Pei Yong Shi, Julien Lescar, Dahai LuoAbstract:Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5' exoribonucleases, ensures efficient expression of viral Proteins, and allows escape from the host innate immune response. The large flavivirus Nonstructural Protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5' RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5' RNA cap: G0pppAG-RNA → (m7)G0pppAG-RNA ("cap-0")→(m7)G0pppAm2'-O-G-RNA ("cap-1"). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 Protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5'-(m7)G0pppA1G2U3U4G5U6U7-3'), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2'-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.
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Characterization of dengue virus resistance to brequinar in cell culture.
Antimicrobial agents and chemotherapy, 2010Co-Authors: Min Qing, Hongping Dong, Gang Zou, Qing Yin Wang, Zhiming Yuan, Pei Yong ShiAbstract:Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope Protein and another mutation (E802Q) located at the priming loop of the Nonstructural Protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope Protein mutation reduced the efficiency of virion assembly/ release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/ release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.
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Flavivirus methyltransferase: A novel antiviral target
Antiviral research, 2008Co-Authors: Hongping Dong, Bo Zhang, Pei Yong ShiAbstract:Abstract Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2′-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of Nonstructural Protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5′ end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m 7 GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA → m 7 GpppA-RNA → m 7 GpppAm-RNA, using S -adenosyl- l -methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2′-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2′-O methylation. In the context of virus, West Nile viruses (WNVs) defective in N-7 methylation are non-replicative; however, WNVs defective in 2′-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.
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Distinct RNA elements confer specificity to flavivirus RNA cap methylation events
Journal of virology, 2007Co-Authors: Hongping Dong, Debashish Ray, Suping Ren, Bo Zhang, Francese Puig-basagoiti, Yuko Takagi, Pei Yong ShiAbstract:The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that Nonstructural Protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
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west nile virus 5 cap structure is formed by sequential guanine n 7 and ribose 2 o methylations by Nonstructural Protein 5
Journal of Virology, 2006Co-Authors: Debashish Ray, Pei Yong Shi, Aaloki Shah, Mark Tilgner, Yi Guo, Yiwei Zhao, Hongping Dong, Tia S Deas, Yangsheng ZhouAbstract:Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the Nonstructural Protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 Protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
David A. Jans - One of the best experts on this subject based on the ideXlab platform.
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the broad spectrum antiviral ivermectin targets the host nuclear transport importin α β1 heterodimer
Antiviral Research, 2020Co-Authors: Sundy N Y Yang, Chunxiao Wang, Sarah C. Atkinson, Natalie A. Borg, Marie A Bogoyevitch, David A. JansAbstract:Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV Nonstructural Protein 5 (NS5) in limiting the host antiviral response. We previously identified the small molecule ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/β1 nuclear transport Proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissociate the preformed IMPα/β1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quantitative bimolecular fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (μM) concentrations. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
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The broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β1 heterodimer
Antiviral research, 2020Co-Authors: Sundy N Y Yang, Chunxiao Wang, Sarah C. Atkinson, Natalie A. Borg, Marie A Bogoyevitch, Alexander Lee, David A. JansAbstract:Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV Nonstructural Protein 5 (NS5) in limiting the host antiviral response. We previously identified the small molecule ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/β1 nuclear transport Proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissociate the preformed IMPα/β1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quantitative bimolecular fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (μM) concentrations. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
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Novel Flavivirus Antiviral That Targets the Host Nuclear Transport Importin α/β1 Heterodimer
Cells, 2019Co-Authors: Sundy N Y Yang, Johanna E. Fraser, Chunxiao Wang, Kylie M. Wagstaff, Jade K Forwood, Sarah C. Atkinson, Belinda Maher, Noelia Roman, Natalie A. Borg, David A. JansAbstract:Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV Nonstructural Protein 5 (NS5) through the action of the host importin (IMP) α/β1 Proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5–IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.
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Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells.
ACS infectious diseases, 2019Co-Authors: Kitti Wing Ki Chan, Wuan Geok Saw, Kate Smith, Satoru Watanabe, Min Jie Alvin Tan, Chin Piaw Gwee, Sarah J. Jeffress, Crystall M. D. Swarbrick, David A. JansAbstract:The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barre syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral Nonstructural Protein 5 (NS5), the most conserved Protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390KRPR393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390ARPA393 mutant Protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-β, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.
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Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin‑α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells
2019Co-Authors: Kitti Wing Ki Chan, Wuan Geok Saw, Satoru Watanabe, Min Jie Alvin Tan, Chin Piaw Gwee, Sarah J. Jeffress, Crystall M. D. Swarbrick, Kate M. Smith, David A. JansAbstract:The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral Nonstructural Protein 5 (NS5), the most conserved Protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390KRPR393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390ARPA393 mutant Protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-β, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5’s nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection
Rob Striker - One of the best experts on this subject based on the ideXlab platform.
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West Nile virus methyltransferase domain interacts with Protein kinase G
Virology Journal, 2013Co-Authors: Julie A. Keating, Dipankar Bhattacharya, Bernard Weisblum, Kristen A Bernard, Pei-yin Lim, Shaun Falk, Mayuri Sharma, Richard J Kuhn, Rob StrikerAbstract:Background The flaviviral Nonstructural Protein 5 (NS5) is a phosphoProtein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent Protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study Protein-Protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro . Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro . While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication.
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Mosquito Protein Kinase G Phosphorylates Flavivirus NS5 and Alters Flight Behavior in Aedes aegypti and Anopheles gambiae
Vector borne and zoonotic diseases (Larchmont N.Y.), 2013Co-Authors: Julie A. Keating, Dipankar Bhattacharya, Samuel S. C. Rund, Spencer Hoover, Ranjit Dasgupta, Samuel J. Lee, Giles E. Duffield, Rob StrikerAbstract:Abstract Many arboviral Proteins are phosphorylated in infected mammalian cells, but it is unknown if the same phosphorylation events occur when insects are similarly infected. One of the mammalian kinases responsible for phosphorylation, Protein kinase G (PKG), has been implicated in the behavior of multiple nonvector insects, but is unstudied in mosquitoes. PKG from Aedes aegypti was cloned, and phosphorylation of specific viral sites was monitored by mass spectrometry from biochemical and cell culture experiments. PKG from Aedes mosquitoes is able to phosphorylate dengue Nonstructural Protein 5 (NS5) at specific sites in cell culture and cell-free systems and autophosphorylates its own regulatory domain in a cell-free system. Injecting Aedes aegypti and Anopheles gambiae mosquitoes with a pharmacological PKG activator resulted in increased Aedes wing activity during periods of their natural diurnal/crepuscular activity and increased Anopheles nocturnal locomotor/flight activity. Thus, perturbation of t...
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Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5
Journal of virology, 2009Co-Authors: Dipankar Bhattacharya, Richard Kuhn, Sonja M. Best, Mayuri, Rushika Perera, Rob StrikerAbstract:Serine/threonine phosphorylation of the Nonstructural Protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 Proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a Protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.
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The flaviviral methyltransferase is a substrate of Casein Kinase 1.
Virus Research, 2009Co-Authors: Dipankar Bhattacharya, Israr-ul H. Ansari, Rob StrikerAbstract:Abstract Serine/Threonine phosphorylation of the Nonstructural Protein 5 (NS5) is a conserved feature of flaviviruses, but the identity and function(s) of the responsible kinase(s) remain unknown. Serine 56 in the methyltransferase domain of NS5 can be phosphorylated intracellularly, is conserved in all flaviviruses, and is a critical residue in the catalytic mechanism. A negative charge at this residue inactivates the 2′-0 methyltransferase activity necessary to form a 5′ cap structure of the viral RNA. Here we show pharmacologic inhibition of Casein Kinase 1 (CK1) suppresses yellow fever virus (YFV) production. We also demonstrate the alpha isoform of Casein Kinase 1 (CK1α), a kinase previously identified as phosphorylating Hepatitis C Virus NS5A Protein, also phosphorylates serine 56 of YFV methyltransferase. Overall these results suggest CK1 activity can influence flaviviral replication.
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Phosphorylation of Yellow Fever Virus NS5 alters methyltransferase activity
Virology, 2008Co-Authors: Dipankar Bhattacharya, Spencer Hoover, Shaun P. Falk, Bernard Weisblum, Martha M. Vestling, Rob StrikerAbstract:Serine/threonine phosphorylation of the Nonstructural Protein 5 (NS5) is conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to characterize phosphorylated residues of yellow fever virus (YFV) NS5 expressed in mammalian cells. Multiple different phosphopeptides were detected. Mutational and additional mass spectrometry data implicated serine 56 (S56), a conserved residue near the active site in the NS5 methyltransferase domain, as one of the phosphorylation sites. Methyltransferase activity is required to form a methylated RNA cap structure and for translation of the YFV polyProtein. We show the 2'-O methylation reaction requires the hydroxyl side chain of S56, and replacement with a negative charge inhibits enzymatic activity. Furthermore mutational alteration of S56, S56A or S56D, prevents amplification in a viral replicon system. Collectively our data suggest phosphorylation of NS5 S56 may act to shut down capping in the viral life cycle.
Hongping Dong - One of the best experts on this subject based on the ideXlab platform.
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Characterization of dengue virus resistance to brequinar in cell culture.
Antimicrobial agents and chemotherapy, 2010Co-Authors: Min Qing, Hongping Dong, Gang Zou, Qing Yin Wang, Zhiming Yuan, Pei Yong ShiAbstract:Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope Protein and another mutation (E802Q) located at the priming loop of the Nonstructural Protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope Protein mutation reduced the efficiency of virion assembly/ release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/ release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.
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Flavivirus methyltransferase: A novel antiviral target
Antiviral research, 2008Co-Authors: Hongping Dong, Bo Zhang, Pei Yong ShiAbstract:Abstract Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2′-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of Nonstructural Protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5′ end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m 7 GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA → m 7 GpppA-RNA → m 7 GpppAm-RNA, using S -adenosyl- l -methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2′-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2′-O methylation. In the context of virus, West Nile viruses (WNVs) defective in N-7 methylation are non-replicative; however, WNVs defective in 2′-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.
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Distinct RNA elements confer specificity to flavivirus RNA cap methylation events
Journal of virology, 2007Co-Authors: Hongping Dong, Debashish Ray, Suping Ren, Bo Zhang, Francese Puig-basagoiti, Yuko Takagi, Pei Yong ShiAbstract:The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that Nonstructural Protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
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west nile virus 5 cap structure is formed by sequential guanine n 7 and ribose 2 o methylations by Nonstructural Protein 5
Journal of Virology, 2006Co-Authors: Debashish Ray, Pei Yong Shi, Aaloki Shah, Mark Tilgner, Yi Guo, Yiwei Zhao, Hongping Dong, Tia S Deas, Yangsheng ZhouAbstract:Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the Nonstructural Protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 Protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
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West Nile Virus 5′-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2′-O Methylations by Nonstructural Protein 5
Journal of virology, 2006Co-Authors: Debashish Ray, Aaloki Shah, Mark Tilgner, Yi Guo, Yiwei Zhao, Hongping Dong, Tia S Deas, Yangsheng Zhou, Pei Yong ShiAbstract:Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the Nonstructural Protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 Protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
C. Cheng Kao - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of the Zika virus full-length NS5 Protein
Nature Communications, 2017Co-Authors: Baoyu Zhao, Yin-chih Chuang, Robert C. Vaughan, Banumathi Sankaran, C. Cheng KaoAbstract:Zika virus infection can cause human birth defects and Guillain-Barré syndrome. Here the authors present the structures of the full-length Nonstructural Protein 5 and its RNA-dependent RNA polymerase domain of Zika virus, which are targets for inhibitors of virus replication. The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication Proteins. Nonstructural Protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 Protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro . Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.
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Structure and Function of the Zika Virus Full-Length NS5 Protein
Nature communications, 2017Co-Authors: Baoyu Zhao, Yin-chih Chuang, Robert C. Vaughan, Banumathi Sankaran, C. Cheng KaoAbstract:The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication Proteins. Nonstructural Protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 A resolution. The NS5 structure has striking similarities to the NS5 Protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.
