83 results on '"Hongping Dong"'
Search Results
2. Solvent-free coating of crosslinked and hydrophobic lignin-based biocomposite for slow-release fertilizer
- Author
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Quan Wei, Lin Zhang, Jienan Chen, Zhaohui Tong, Xiaoxun Zhou, Lishu Shao, Zhiping Wu, Peng Zhan, Fen Wang, Na Liu, Hanxue Lin, and Hongping Dong
- Subjects
Hydrophobic sodium lignosulphonate ,Tg-adjustable ,Solvent-free coating technology ,Slow-release fertilizer ,Polymers and polymer manufacture ,TP1080-1185 - Abstract
Biopolymer-based slow-release fertilizers (SRFs) have attracted increasing interest because of their environmental benefits. Herein, a hydrophobic thermoplastic lignin with favorable film formation properties was synthesized through simple crosslinking followed by esterification reactions. The chemical structure, glass transition temperature, and hydrophobicity were determined using Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, differential scanning calorimetry, and contact angle studies. The newly synthesized esterified crosslinked sodium lignosulfonate (ECSL) enables the encapsulation of urea via a solvent-free coating method to prepare SRFs because of its appropriate glass transition temperature (Tg) and excellent film formation properties. The ECSL-based SRF released only 86.9% of the encapsulated urea within 44 d, which is superior to most lignin-based SRFs in previous studies. The release rate can also be tuned by adjusting the ECSL ratio. The synthesis of hydrophobic lignin biocomposites with suitable Tgs for film formation resolves the bottleneck of using these types of non-thermoplastic polymers (e.g., lignin) for SRFs. This study not only maximizes the value of biowaste lignin but also offers a non-paradigm approach toward low-cost SRFs for sustainable agriculture.
- Published
- 2021
- Full Text
- View/download PDF
3. The Natural Product Cavinafungin Selectively Interferes with Zika and Dengue Virus Replication by Inhibition of the Host Signal Peptidase
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David Estoppey, Chia Min Lee, Marco Janoschke, Boon Heng Lee, Kah Fei Wan, Hongping Dong, Philippe Mathys, Ireos Filipuzzi, Tim Schuhmann, Ralph Riedl, Thomas Aust, Olaf Galuba, Gregory McAllister, Carsten Russ, Martin Spiess, Tewis Bouwmeester, Ghislain M.C. Bonamy, and Dominic Hoepfner
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses. : Recent outbreaks and lack of effective treatments against dengue and Zika virus have caused public concerns. Estoppey et al. have identified cavinafungin as exerting potent and selective antiviral activity by targeting the signal-binding cleft of the catalytic subunit of the endoplasmic reticulum signal peptidase. Keywords: Zika virus, dengue virus, cavinafungin, signal peptidase, SEC11A, SEC11, CRISPR/Cas9, chemogenomic profiling
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- 2017
- Full Text
- View/download PDF
4. Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C.
- Author
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Xin-Ni Lim, Chao Shan, Jan K Marzinek, Hongping Dong, Thiam Seng Ng, Justin S G Ooi, Guntur Fibriansah, Jiaqi Wang, Chandra S Verma, Peter J Bond, Pei-Yong Shi, and Shee-Mei Lok
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.
- Published
- 2019
- Full Text
- View/download PDF
5. Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants.
- Author
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Roland Züst, Shi-Hua Li, Xuping Xie, Sumathy Velumani, Melissa Chng, Ying-Xiu Toh, Jing Zou, Hongping Dong, Chao Shan, Jassia Pang, Cheng-Feng Qin, Evan W Newell, Pei-Yong Shi, and Katja Fink
- Subjects
Medicine ,Science - Abstract
Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500'000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2'-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2'-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2'-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.
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- 2018
- Full Text
- View/download PDF
6. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.
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Siew Pheng Lim, Christian Guy Noble, Cheah Chen Seh, Tingjin Sherryl Soh, Abbas El Sahili, Grace Kar Yarn Chan, Julien Lescar, Rishi Arora, Timothy Benson, Shahul Nilar, Ujjini Manjunatha, Kah Fei Wan, Hongping Dong, Xuping Xie, Pei-Yong Shi, and Fumiaki Yokokawa
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
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.
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- 2016
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7. Correction: Rational Design of a Live Attenuated Dengue Vaccine: 2′--Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques.
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Roland Züst, Hongping Dong, Xiao-Feng Li, David C. Chang, Bo Zhang, Thavamalar Balakrishnan, Ying-Xiu Toh, Tao Jiang, Shi-Hua Li, Yong-Qiang Deng, Brett R. Ellis, Esther M. Ellis, Michael Poidinger, Francesca Zolezzi, Cheng-Feng Qin, Pei-Yong Shi, and Katja Fink
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2013
- Full Text
- View/download PDF
8. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.
- Author
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Roland Züst, Hongping Dong, Xiao-Feng Li, David C Chang, Bo Zhang, Thavamalar Balakrishnan, Ying-Xiu Toh, Tao Jiang, Shi-Hua Li, Yong-Qiang Deng, Brett R Ellis, Esther M Ellis, Michael Poidinger, Francesca Zolezzi, Cheng-Feng Qin, Pei-Yong Shi, and Katja Fink
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.
- Published
- 2013
- Full Text
- View/download PDF
9. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.
- Author
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Hongping Dong, David C Chang, Maggie Ho Chia Hua, Siew Pheng Lim, Yok Hian Chionh, Fabian Hia, Yie Hou Lee, Petra Kukkaro, Shee-Mei Lok, Peter C Dedon, and Pei-Yong Shi
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.
- Published
- 2012
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10. Table S1, S2, S3 and S4; Figure S1, S2,and S3 from An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor Progression by Modulating the Immune Response
- Author
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Bin Zou, Hailong Zhang, Yanchao Liu, Xianlei Fu, Shichao Ma, Yanni Chen, Lijing Kang, Sheng Chen, Xuejie Zhang, Shaojun Liu, and Hongping Dong
- Abstract
Data for article support. Supplementary Table 1. Antibody used for ELISA and WB; Supplementary Table 2. Primers used for quantitative Real-time RT-PCR; Supplementary Table 3. BR-A selectivity across a panel of kinases; Supplementary Table 4. EED-BR-001 Data collection and refinement statistics; Supplementary Figure 1. BR-001 decreases global H3K27 methylation. Inhibition of H3K27 methylation in cells treated with different concentrations of BR-001 was measured. KATO III and SNU16 cells were treated with BR-001 for 3 days at the indicated concentrations. Levels of H3K27me3 in cell lysate was detected using ELISA . All experiments were repeated three times and representative data are shown; Supplementary Figure 2. BR-001 suppress tumor growth in mouse xenograft and syngeneic models. Effect of BR-001 on body weight in Karpas422 xenograft (A), Pfeiffer xenograft (B) and murine CT26 models (C) (n=10). The data are shown as mean {plus minus} SEM; Supplementary Figure 3. Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs). Counts of MDSCs (A), NK cells (B), TAMs (C) , CD45+ cells (D), CD3+ cells (E) and CD4+ cells (F) in fresh tumors collected at the last day of BR-001 dosing (Figure 4D, n=10). The data are shown as mean {plus minus} SEM
- Published
- 2023
11. Data from An Allosteric PRC2 Inhibitor Targeting EED Suppresses Tumor Progression by Modulating the Immune Response
- Author
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Bin Zou, Hailong Zhang, Yanchao Liu, Xianlei Fu, Shichao Ma, Yanni Chen, Lijing Kang, Sheng Chen, Xuejie Zhang, Shaojun Liu, and Hongping Dong
- Abstract
Aberrant activity of polycomb repressive complex 2 (PRC2) is involved in a wide range of human cancer progression. The WD40 repeat-containing protein EED is a core component of PRC2 and enhances PRC2 activity through interaction with H3K27me3. In this study, we report the discovery of a class of pyrimidone compounds, represented by BR-001, as potent allosteric inhibitors of PRC2. X-ray co-crystallography showed that BR-001 directly binds EED in the H3K27me3-binding pocket. BR-001 displayed antitumor potency in vitro and in vivo. In Karpas422 and Pfeiffer xenograft mouse models, twice daily oral dosing with BR-001 resulted in robust antitumor activity. BR-001 was also efficacious in syngeneic CT26 colon tumor-bearing mice; oral dosing of 30 mg/kg of BR-001 led to 59.3% tumor growth suppression and increased frequency of effector CD8+ T-cell infiltrates in tumors. Pharmacodynamic analysis revealed that CXCL10 was highly upregulated, suggesting that CXCL10 triggers the trafficking of CD8+ T cells toward tumor sites. Our results demonstrate for the first time that inhibition of EED modulates the tumor immune microenvironment to induce regression of colon tumors and therefore has the potential to be used in combination with immune-oncology therapy.Significance:BR-001, a potent inhibitor of the EED subunit of the PRC2 complex, suppresses tumor progression by modulating the tumor microenvironment.
- Published
- 2023
12. Wood-derived bio-coating materials incorporating hydrophobic lignin and hierarchically porous biochar for high-efficiency coating slow-release fertilizers
- Author
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Hongping Dong, Shifeng Tang, Lin Zhang, Zhaohui Tong, Zhiping Wu, Peng Zhan, Lishu Shao, Yan Qing, and Jin Liu
- Subjects
Structural Biology ,General Medicine ,Molecular Biology ,Biochemistry - Published
- 2023
13. No-reference image quality assessment in curvelet domain.
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Lixiong Liu, Hongping Dong, Hua Huang 0001, and Alan C. Bovik
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- 2014
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14. Solvent-free coating of crosslinked and hydrophobic lignin-based biocomposite for slow-release fertilizer
- Author
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Jienan Chen, Zhaohui Tong, Quan Wei, Zhiping Wu, Peng Zhan, Na Liu, Lin Zhang, Fen Wang, Hanxue Lin, Hongping Dong, Xiaoxun Zhou, and Lishu Shao
- Subjects
chemistry.chemical_classification ,Materials science ,Polymers and Plastics ,Sodium lignosulfonate ,Organic Chemistry ,Tg-adjustable ,Solvent-free coating technology ,Slow-release fertilizer ,Hydrophobic sodium lignosulphonate ,Polymer ,engineering.material ,chemistry.chemical_compound ,Differential scanning calorimetry ,TP1080-1185 ,Chemical engineering ,chemistry ,engineering ,Lignin ,Biopolymer ,Polymers and polymer manufacture ,Fourier transform infrared spectroscopy ,Biocomposite ,Glass transition - Abstract
Biopolymer-based slow-release fertilizers (SRFs) have attracted increasing interest because of their environmental benefits. Herein, a hydrophobic thermoplastic lignin with favorable film formation properties was synthesized through simple crosslinking followed by esterification reactions. The chemical structure, glass transition temperature, and hydrophobicity were determined using Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, differential scanning calorimetry, and contact angle studies. The newly synthesized esterified crosslinked sodium lignosulfonate (ECSL) enables the encapsulation of urea via a solvent-free coating method to prepare SRFs because of its appropriate glass transition temperature (Tg) and excellent film formation properties. The ECSL-based SRF released only 86.9% of the encapsulated urea within 44 d, which is superior to most lignin-based SRFs in previous studies. The release rate can also be tuned by adjusting the ECSL ratio. The synthesis of hydrophobic lignin biocomposites with suitable Tgs for film formation resolves the bottleneck of using these types of non-thermoplastic polymers (e.g., lignin) for SRFs. This study not only maximizes the value of biowaste lignin but also offers a non-paradigm approach toward low-cost SRFs for sustainable agriculture.
- Published
- 2021
15. NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models
- Author
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Haoying Xu, Wei Lin Sandra Sim, Cheah Chen Seh, Feng Wang, Thierry T. Diagana, Wai Ling Chan, Jae-Geun Song, Kah Fei Wan, Bin Zou, Ghislain M. C. Bonamy, David Beer, David T. Barkan, Min Li, Stephanie A. Moquin, Francesca Blasco, Suresh B. Lakshminarayana, Jin Zhang, Oliver Simon, Vito G. Sasseville, Craig W. Day, Qing-Yin Wang, Chandrassegar Saravanan, Katherine Chan, Fumiaki Yokokawa, Bryan K. S. Yeung, Ratna Karuna, Hui-Quan Yeo, Colin Osborne, Christopher Sarko, Pei Yong Shi, Hongping Dong, Mei Ding, Siew Pheng Lim, Yen Liang Chen, Feng Gu, Cyrille Kounde, Gang Wang, Siyan Lu, and Wei Liu
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0301 basic medicine ,biology ,medicine.drug_class ,business.industry ,030106 microbiology ,Viremia ,General Medicine ,Dengue virus ,Pharmacology ,medicine.disease_cause ,medicine.disease ,biology.organism_classification ,Dengue fever ,Bioavailability ,03 medical and health sciences ,Flavivirus ,030104 developmental biology ,Pharmacokinetics ,medicine ,Potency ,Antiviral drug ,business - Abstract
Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.
- Published
- 2021
16. The Natural Product Cavinafungin Selectively Interferes with Zika and Dengue Virus Replication by Inhibition of the Host Signal Peptidase
- Author
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Chia Min Lee, Kah Fei Wan, Ralph Riedl, Marco Janoschke, Ghislain M. C. Bonamy, Tewis Bouwmeester, Olaf Galuba, Hongping Dong, Philippe Mathys, Tim Schuhmann, David Estoppey, Martin Spiess, Carsten Russ, Dominic Hoepfner, Ireos Filipuzzi, Boon Heng Lee, Gregory McAllister, and Thomas Aust
- Subjects
0301 basic medicine ,Signal peptide ,Protein subunit ,030106 microbiology ,Saccharomyces cerevisiae ,Dengue virus ,medicine.disease_cause ,Virus Replication ,General Biochemistry, Genetics and Molecular Biology ,Zika virus ,03 medical and health sciences ,Lipopeptides ,Viral Proteins ,medicine ,CRISPR ,Humans ,Flavivirus Infections ,lcsh:QH301-705.5 ,Signal peptidase ,Biological Products ,biology ,Cas9 ,Genome, Human ,Serine Endopeptidases ,Membrane Proteins ,Genomics ,Zika Virus ,Dengue Virus ,biology.organism_classification ,HCT116 Cells ,Virology ,Protein Subunits ,030104 developmental biology ,lcsh:Biology (General) ,Gene Knockdown Techniques ,CRISPR-Cas Systems - Abstract
Summary: Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses. : Recent outbreaks and lack of effective treatments against dengue and Zika virus have caused public concerns. Estoppey et al. have identified cavinafungin as exerting potent and selective antiviral activity by targeting the signal-binding cleft of the catalytic subunit of the endoplasmic reticulum signal peptidase. Keywords: Zika virus, dengue virus, cavinafungin, signal peptidase, SEC11A, SEC11, CRISPR/Cas9, chemogenomic profiling
- Published
- 2017
17. Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit
- Author
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Hongping Dong, Bryan K. S. Yeung, Ratna Karuna, Bin Zou, Trixie Wagner, Hui-Quan Yeo, Cyrille Kounde, Oliver Simon, Kah Fei Wan, Qing-Yin Wang, Ghislain M. C. Bonamy, Fumiaki Yokokawa, and Ina Dix
- Subjects
0301 basic medicine ,Scaffold ,High-throughput screening ,Clinical Biochemistry ,Pharmaceutical Science ,Dengue virus ,medicine.disease_cause ,Antiviral Agents ,01 natural sciences ,Biochemistry ,Piperazines ,Virus ,Cell Line ,Pyrrolopiperazinone ,Dengue fever ,Structure-Activity Relationship ,03 medical and health sciences ,Drug Discovery ,medicine ,Humans ,Molecular Biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Dengue Virus ,medicine.disease ,Phenotype ,Virology ,High-Throughput Screening Assays ,0104 chemical sciences ,030104 developmental biology ,Molecular Medicine - Abstract
A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50
- Published
- 2017
18. Evasion of early innate immune response by 2′- O -methylation of dengue genomic RNA
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Long T. Hoang, Mark Schreiber, Ahmad Nazri Mohamed Naim, Min Jie Alvin Tan, David C. Chang, Martin L. Hibberd, Hongping Dong, and Pei Yong Shi
- Subjects
Innate immune response ,0301 basic medicine ,NS5 ,viruses ,Dengue virus ,Biology ,medicine.disease_cause ,Article ,Virus ,Cell Line ,Dengue fever ,Dengue ,03 medical and health sciences ,Immune system ,Interferon ,Virology ,medicine ,Humans ,Methyltransferase ,Immune Evasion ,Innate immune system ,Point mutation ,virus diseases ,RNA ,Genomics ,DNA Methylation ,Dengue Virus ,biochemical phenomena, metabolism, and nutrition ,medicine.disease ,Immunity, Innate ,030104 developmental biology ,RNA, Viral ,medicine.drug - Abstract
Dengue virus (DENV) is the most prevalent mosquito-borne virus pathogen in humans. There is currently no antiviral therapeutic or widely available vaccine against dengue infection. The DENV RNA genome is methylated on its 5′ cap by its NS5 protein. DENV bearing a single E216A point mutation in NS5 loses 2′-O-methylation of its genome. While this mutant DENV is highly attenuated and immunogenic, the mechanism of this attenuation has not been elucidated. In this study, we find that replication of this mutant DENV is attenuated very early during infection. This early attenuation is not dependent on a functional type I interferon response and coincides with early activation of the innate immune response. Taken together, our data suggest that 2′-O-methylation of DENV genomic RNA is important for evasion of the host immune response during the very early stages of infection as the virus seeks to establish infection., Highlights • Dengue virus lacking 2′-O-methylation of its genome is attenuated and immunogenic. • This mutant virus is attenuated during the early stages of infection. • 2′-O-methylation of virus RNA is important for evading the innate immune response.
- Published
- 2016
19. Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C
- Author
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Jiaqi Wang, Chandra S. Verma, Xin Ni Lim, Pei Yong Shi, Justin S.G. Ooi, Peter J. Bond, Chao Shan, Guntur Fibriansah, Thiam Seng Ng, Jan K. Marzinek, Shee-Mei Lok, Hongping Dong, School of Biological Sciences, and Bioinformatics Institute, A*STAR
- Subjects
Models, Molecular ,RNA viruses ,Physiology ,Thermal Stability ,Clone (cell biology) ,Dengue virus ,Pathology and Laboratory Medicine ,medicine.disease_cause ,Biochemistry ,Database and Informatics Methods ,Mathematical and Statistical Techniques ,Viral Envelope Proteins ,Immune Physiology ,Medicine and Health Sciences ,Biochemical Simulations ,Biology (General) ,Antigens, Viral ,Peptide sequence ,Principal Component Analysis ,0303 health sciences ,Mutation ,Immune System Proteins ,Chemistry ,Physics ,Microbial Mutation ,Statistics ,030302 biochemistry & molecular biology ,Temperature ,Biological sciences [Science] ,DENV2 ,3. Good health ,Medical Microbiology ,Viral Pathogens ,Viruses ,Physical Sciences ,Thermodynamics ,Pathogens ,Sequence Analysis ,Research Article ,Bioinformatics ,QH301-705.5 ,Immunology ,Sequence alignment ,Molecular Dynamics Simulation ,Serogroup ,Research and Analysis Methods ,Microbiology ,Antibodies ,Virus ,Cell Line ,Protein–protein interaction ,03 medical and health sciences ,Antigen ,Virology ,Genetics ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Statistical Methods ,Protein Interactions ,Microbial Pathogens ,Molecular Biology ,030304 developmental biology ,Sequence Homology, Amino Acid ,Flaviviruses ,Cryoelectron Microscopy ,Organisms ,Biology and Life Sciences ,Proteins ,Computational Biology ,Dengue Virus ,RC581-607 ,Amino Acid Substitution ,Multivariate Analysis ,Biophysics ,Parasitology ,Immunologic diseases. Allergy ,Sequence Alignment ,Mathematics - Abstract
The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to “bumpy” surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become “bumpy”. These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus., Author summary DENV2 particles have been shown to change their morphologies (compact smooth to loose bumpy surfaced) when temperature is switched from 28°C to 37°C. We used two DENV2 viruses both belonging to the same strain designation but with a different passage history—one of which exhibited the smooth surfaced morphology while the other was bumpy surfaced, observed by cryoEM. We mutated residues in the E protein of the DENV2 infectious clone that has the smooth surfaced morphology to determine if any could result in a bumpy morphology. Results showed several different mutations could lead to this change. Using molecular dynamics simulations, we showed how these mutations likely destabilize the E protein dimeric interactions. We investigated whether the bumpy morphology also occurs in DENV2 clinical isolates, and showed that these viruses can exhibit both morphologies, indicating that vaccine and therapeutics development should target both virus forms.
- Published
- 2019
20. Dengue NS2A Protein Orchestrates Virus Assembly
- Author
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CongBao Kang, Jing Zou, Vsevolod L. Popov, Xuping Xie, Qing Yin Wang, Hongping Dong, Andrew Routh, Pei Yong Shi, Yiyang Zhou, Xianwen Zhang, and Xinwen Chen
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Untranslated region ,viruses ,Dengue virus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,03 medical and health sciences ,Viral Proteins ,0302 clinical medicine ,Viral Envelope Proteins ,Aedes ,Virology ,Cricetinae ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Nucleocapsid ,Vero Cells ,030304 developmental biology ,0303 health sciences ,NS3 ,biology ,Virus Assembly ,Serine Endopeptidases ,virus diseases ,RNA ,biochemical phenomena, metabolism, and nutrition ,Dengue Virus ,biology.organism_classification ,humanities ,Cell biology ,Flavivirus ,HEK293 Cells ,Capsid ,Virion assembly ,RNA, Viral ,Parasitology ,030217 neurology & neurosurgery ,RNA Helicases - Abstract
Dengue virus assembly requires cleavage of viral C-prM-E polyprotein into three structural proteins (capsid, premembrane, and envelope), packaging of viral RNA with C protein into nucleocapsid, and budding of prM and E proteins into virions. The molecular mechanisms underlying these assembly events are unclear. Here, we show that dengue nonstructural protein 2A (NS2A protein) recruits viral RNA, structural proteins, and protease to the site of virion assembly and coordinates nucleocapsid and virus formation. The last 285 nucleotides of viral 3' UTR serve as a "recruiting signal for packaging" that binds to a cytosolic loop of NS2A. This interaction allows NS2A to recruit nascent RNA from the replication complex to the virion assembly site. NS2A also recruits the C-prM-E polyprotein and NS2B-NS3 protease to the virion assembly site by interacting with prM, E, and NS3, leading to coordinated C-prM-E cleavage. Mature C protein assembles onto genomic RNA to form nucleocapsid, followed by prM and E envelopment and virion formation.
- Published
- 2019
21. Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA
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Weiling Maggie Cai, Jin Wang, Zhenguo Lin, Liang Cui, Hongping Dong, Luang Xu, Samie R. Jaffrey, Timothy K. Lu, Seetharamsingh Balamkundu, Dahai Luo, Peter C. Dedon, Chuan-Fa Liu, Bing Liang Alvin Chew, Yong Lai, Pei Yong Shi, Xin-Yuan Fu, School of Biological Sciences, Interdisciplinary Graduate School (IGS), Lee Kong Chian School of Medicine (LKCMedicine), and NTU Institute of Health Technologies
- Subjects
RNA Caps ,Sequence analysis ,Mass Spectrometry-based Technique ,Metabolite ,Saccharomyces cerevisiae ,Biology ,Dengue virus ,medicine.disease_cause ,RNA Characterisation and Manipulation ,Epigenesis, Genetic ,Transcriptome ,03 medical and health sciences ,Mice ,chemistry.chemical_compound ,Genetics ,medicine ,Animals ,Humans ,Medicine [Science] ,Nucleotide ,RNA Processing, Post-Transcriptional ,Escherichia coli ,Cells, Cultured ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Sequence Analysis, RNA ,030302 biochemistry & molecular biology ,Eukaryotic transcription ,RNA ,Methylation ,Dengue Virus ,Yeast ,Mice, Inbred C57BL ,carbohydrates (lipids) ,chemistry ,Biochemistry ,Methods Online ,RNA, Viral ,Female - Abstract
Chemical modification of transcripts with 5’ caps occurs in all organisms. Here we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of the cap epitranscriptome in any organism. The method was piloted with 21 canonical caps – m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG – and 5 “metabolite” caps – NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus,Escherichia coli, yeast, mice, and humans, we discovered four new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and surprisingly high proportions of caps lacking 2’-O-methylation, such as m7Gpppm6A in mammals and m7GpppA in dengue virus, and we did not detect cap m1A/m1Am in humans. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps. The mystery around cap m1A/m1Am analysis remains unresolved.
- Published
- 2019
22. The role of sequence context, nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA
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Sasilada Sirirungruang, Peter C. Dedon, Jin Wang, Hongping Dong, Yok Hian Chionh, Richard P. Cunningham, Megan E. McBee, Pei Yong Shi, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, and Dedon, Peter C
- Subjects
0301 basic medicine ,Deoxyribonucleotides ,RNA-dependent RNA polymerase ,Biology ,Cell Line ,03 medical and health sciences ,Stress, Physiological ,Tandem Mass Spectrometry ,Genetics ,Animals ,Humans ,Mammals ,Base Composition ,Hydrolysis ,Intron ,RNA ,Ribonucleotides ,Non-coding RNA ,3. Good health ,Antisense RNA ,RNA, Bacterial ,RNA silencing ,Eukaryotic Cells ,030104 developmental biology ,Prokaryotic Cells ,Mutagenesis ,RNA editing ,RNA, Viral ,Small nuclear RNA ,Chromatography, Liquid - Abstract
The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10[superscript 3] to 10[superscript 5] ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections., National Institutes of Health (U.S.) (Grant ES022858), Singapore. National Research Foundation, Singapore-MIT Alliance for Research and Technology
- Published
- 2016
23. Discovery of Dengue Virus NS4B Inhibitors
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Julien Lescar, Feng Gu, Kah Fei Wan, Francesca Blasco, CongBao Kang, Wai Ling Chan, Wei Liu, Paul W. Smith, Agatha Susila, Bin Zou, Qing Yin Wang, K.L. Yeo, Mei Ding, Chao Shan, Andy M. Yip, Haoying Xu, Hongping Dong, Jing Zou, Suresh B. Lakshminarayana, Ratna Karuna, Pei Yong Shi, Peck Gee Seah, Diamond, M. S., and School of Biological Sciences
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viruses ,Immunology ,Viremia ,Viral Nonstructural Proteins ,Dengue virus ,Biology ,medicine.disease_cause ,Antiviral Agents ,Microbiology ,Cell Line ,In vivo ,Cricetinae ,Virology ,Vaccines and Antiviral Agents ,Drug Discovery ,medicine ,Animals ,Humans ,Spiro Compounds ,Replicon ,chemistry.chemical_classification ,Drug discovery ,virus diseases ,Dengue Virus ,biochemical phenomena, metabolism, and nutrition ,medicine.disease ,In vitro ,Amino acid ,chemistry ,Insect Science ,Viral replication complex - Abstract
The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo . The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC 50 ], 10 to 80 nM) but not DENV-1 and -4 (EC 50 , >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo . Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.
- Published
- 2015
24. Characterization of Dengue Virus NS4A and NS4B Protein Interaction
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CongBao Kang, Zhiming Yuan, Qing Yin Wang, Xuping Xie, Michelle Yueqi Lee, Hongping Dong, Pei Yong Shi, and Jing Zou
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Magnetic Resonance Spectroscopy ,Protein Conformation ,viruses ,DNA Mutational Analysis ,Immunology ,Plasma protein binding ,Viral Nonstructural Proteins ,Biology ,Dengue virus ,Virus Replication ,medicine.disease_cause ,Microbiology ,Cell Line ,Protein structure ,Cricetinae ,Virology ,Protein Interaction Mapping ,medicine ,Animals ,Humans ,Protein secondary structure ,chemistry.chemical_classification ,Structure and Assembly ,Endoplasmic reticulum ,Dengue Virus ,Recombinant Proteins ,Amino acid ,Transmembrane domain ,Viral replication ,Biochemistry ,chemistry ,Insect Science ,Protein Binding - Abstract
Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated K d (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach. IMPORTANCE Flavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.
- Published
- 2015
25. Lead Optimization of Spiropyrazolopyridones: A New and Potent Class of Dengue Virus Inhibitors
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Hao Ying Xu, Wei Liu, Feng Gu, Bin Zou, Wai Ling Chan, Paul W. Smith, Kah Fei Wan, Ratna Karuna, Agatha Susila, Peck Gee Seah, Andy Yip, Pei Yong Shi, Trixie Wagner, Thierry T. Diagana, Qing Yin Wang, Alex Chao, Mei Ding, Hongping Dong, Seh Yong Leong, Francesca Blasco, Katherine Chan, Shahul Nilar, and Ina Dix
- Subjects
Organic Chemistry ,virus diseases ,Viremia ,Biology ,Dengue virus ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Virology ,Dengue fever ,Pharmacokinetics ,In vivo ,Drug Discovery ,medicine ,Structure–activity relationship ,Potency ,Enantiomer - Abstract
Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model.
- Published
- 2015
26. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine
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Shihua Li, Hongping Dong, Pei Yong Shi, Christian G. Noble, and Sock Hui Chew
- Subjects
Models, Molecular ,S-Adenosylmethionine ,Methyltransferase ,viruses ,Structure-based design ,Dengue virus ,Crystallography, X-Ray ,medicine.disease_cause ,Article ,law.invention ,Viral Proteins ,Sinefungin ,law ,Virology ,medicine ,Transferase ,Protein Footprinting ,Pharmacology ,chemistry.chemical_classification ,Binding Sites ,biology ,Flavivirus ,Crystal structure ,Methyltransferases ,Methylation ,Dengue Virus ,biology.organism_classification ,Enzyme ,chemistry ,Biochemistry ,Recombinant DNA ,Protein Binding - Abstract
Highlights • A method to produce the dengue methyltransferase without a bound SAM is presented. • The enzyme retains enzymatic activity. • The high-resolution crystal structure of the free MTase shows there is no conformational change on SAM binding. • This allows rational drug discovery of compounds that compete with SAM binding. • This is demonstrated by solving the crystal structure of DENV MTase bound to Sinefungin., Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2′-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.
- Published
- 2014
27. Flavivirus RNA methylation
- Author
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Cheng-Feng Qin, Hongping Dong, Roland Züst, Katja Fink, Siew Pheng Lim, and Pei Yong Shi
- Subjects
Genetics ,tRNA Methyltransferases ,biology ,Picornavirus ,Flavivirus ,viruses ,Viral translation ,RNA ,Dengue virus ,RNA Cap Analogs ,biology.organism_classification ,medicine.disease_cause ,Methylation ,Virology ,Internal ribosome entry site ,Viral entry ,Protein Biosynthesis ,Viral evolution ,medicine ,RNA, Viral ,RNA Processing, Post-Transcriptional ,Immune Evasion - Abstract
The 5′ end of eukaryotic mRNA contains the type-1 (m7GpppNm) or type-2 (m7GpppNmNm) cap structure. Many viruses have evolved various mechanisms to develop their own capping enzymes (e.g. flavivirus and coronavirus) or to ‘steal’ caps from host mRNAs (e.g. influenza virus). Other viruses have developed ‘cap-mimicking’ mechanisms by attaching a peptide to the 5′ end of viral RNA (e.g. picornavirus and calicivirus) or by having a complex 5′ RNA structure (internal ribosome entry site) for translation initiation (e.g. picornavirus, pestivirus and hepacivirus). Here we review the diverse viral RNA capping mechanisms. Using flavivirus as a model, we summarize how a single methyltransferase catalyses two distinct N-7 and 2′-O methylations of viral RNA cap in a sequential manner. For antiviral development, a structural feature unique to the flavivirus methyltransferase was successfully used to design selective inhibitors that block viral methyltransferase without affecting host methyltransferases. Functionally, capping is essential for prevention of triphosphate-triggered innate immune activation; N-7 methylation is critical for enhancement of viral translation; and 2′-O methylation is important for subversion of innate immune response during viral infection. Flaviviruses defective in 2′-O methyltransferase are replicative, but their viral RNAs lack 2′-O methylation and are recognized and eliminated by the host immune response. Such mutant viruses could be rationally designed as live attenuated vaccines. This concept has recently been proved with Japanese encephalitis virus and dengue virus. The findings obtained with flavivirus should be applicable to other RNA viruses.
- Published
- 2014
28. Dimerization of Flavivirus NS4B Protein
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CongBao Kang, Julien Lescar, Le Tian Lee, Zhiming Yuan, Pei Yong Shi, Aline Reynaud, Ramya Chandrasekaran, Lijian Yap, Qing Yin Wang, Xuping Xie, Hongping Dong, Jing Zou, and School of Biological Sciences
- Subjects
viruses ,Amino Acid Motifs ,Immunology ,Mutagenesis (molecular biology technique) ,Viral Nonstructural Proteins ,Biology ,Dengue virus ,medicine.disease_cause ,Microbiology ,Dengue ,Replication factor C ,Virology ,medicine ,Humans ,Replicon ,Endoplasmic reticulum ,RNA ,Dengue Virus ,Genome Replication and Regulation of Viral Gene Expression ,Science::Biological sciences [DRNTU] ,Membrane protein ,Biochemistry ,Viral replication ,Insect Science ,Dimerization ,West Nile virus ,West Nile Fever - Abstract
Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli , reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro , as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans -Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans -complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans -complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. IMPORTANCE The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans -complementation enhances our understanding of NS4B in flavivirus replication.
- Published
- 2014
29. Ten years of dengue drug discovery: Progress and prospects
- Author
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Yen Liang Chen, Shahul Nilar, Paul Smith, Christian G. Noble, Hongping Dong, Fumiaki Yokokawa, Bin Zou, David Beer, Pei Yong Shi, Julien Lescar, Siew Pheng Lim, and Qing Yin Wang
- Subjects
viruses ,Balapiravir ,Biology ,Dengue virus ,medicine.disease_cause ,Antiviral Agents ,History, 21st Century ,Dengue fever ,Dengue ,chemistry.chemical_compound ,Virology ,Drug Discovery ,medicine ,Humans ,Repurposing ,Pharmacology ,Singapore ,Nucleoside analogue ,Drug discovery ,Dengue Virus ,medicine.disease ,biology.organism_classification ,Clinical trial ,Flavivirus ,chemistry ,medicine.drug - Abstract
To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy.
- Published
- 2013
30. Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′- O Methylation
- Author
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E-De Qin, Yong-Qiang Deng, Xiaofeng Li, Shihua Li, Cheng-Feng Qin, Qibin Leng, Qing Ye, Hui Zhao, Xiaoyu Wang, Xuping Xie, Shun-Ya Zhu, Hong-Jiang Wang, Bo Zhang, Roland Zuest, Pei Yong Shi, and Hongping Dong
- Subjects
viruses ,Immunology ,Biology ,Antibodies, Viral ,Vaccines, Attenuated ,Recombinant virus ,Methylation ,Microbiology ,Virus ,Mice ,Viral entry ,Interferon ,Virology ,Vaccines and Antiviral Agents ,medicine ,Animals ,Encephalitis, Japanese ,Encephalitis Virus, Japanese ,Mice, Inbred BALB C ,tRNA Methyltransferases ,Attenuated vaccine ,Japanese Encephalitis Vaccines ,RNA ,biology.organism_classification ,Survival Analysis ,TRNA Methyltransferases ,Disease Models, Animal ,Flavivirus ,Insect Science ,Leukocytes, Mononuclear ,RNA, Viral ,Female ,medicine.drug - Abstract
Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2′- O cap of their RNA; alternatively, they “snatch” host mRNA cap to form the 5′ end of viral RNA. The function of 2′- O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2′- O methylation is replicative, but its viral RNA lacks 2′- O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2′- O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2′- O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo . A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2′- O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2′- O methyltransferases.
- Published
- 2013
31. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling
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Fumiaki Yokokawa, Kah Fei Wan, Tingjin Sherryl Soh, Julien Lescar, Timothy E. Benson, Shahul Nilar, Ujjini H. Manjunatha, Christian G. Noble, Rishi Arora, Pei Yong Shi, Siew Pheng Lim, Xuping Xie, Abbas El Sahili, Grace Kar Yarn Chan, Cheah Chen Seh, Hongping Dong, Novartis Institute for Tropical Diseases (NITD), Nanyang Technological University [Singapour], Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Novartis Institutes for BioMedical Research (NIBR), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), and HAL UPMC, Gestionnaire
- Subjects
0301 basic medicine ,viruses ,Hands ,Dengue virus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Crystallography, X-Ray ,Biochemistry ,Polymerases ,Dengue ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Medicine and Health Sciences ,Thumbs ,Ribozymes ,lcsh:QH301-705.5 ,Musculoskeletal System ,Polymerase ,[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Crystallography ,biology ,Physics ,Ribozyme ,virus diseases ,Melting ,Condensed Matter Physics ,3. Good health ,Enzymes ,Nucleic acids ,Flavivirus ,RNA silencing ,Arms ,Physical Sciences ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,[SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Crystal Structure ,Anatomy ,Oxidoreductases ,Luciferase ,Phase Transitions ,Research Article ,lcsh:Immunologic diseases. Allergy ,Nucleic acid synthesis ,Immunology ,Allosteric regulation ,RNA-dependent RNA polymerase ,Antiviral Agents ,Microbiology ,03 medical and health sciences ,Protein Domains ,Virology ,DNA-binding proteins ,Genetics ,medicine ,Humans ,Solid State Physics ,Chemical synthesis ,RNA synthesis ,Molecular Biology ,Nucleic Acid Synthesis Inhibitors ,030102 biochemistry & molecular biology ,Biology and life sciences ,Limbs (Anatomy) ,RNA ,Proteins ,biochemical phenomena, metabolism, and nutrition ,Dengue Virus ,Surface Plasmon Resonance ,biology.organism_classification ,RNA-Dependent RNA Polymerase ,Viral Replication ,Research and analysis methods ,Biosynthetic techniques ,030104 developmental biology ,lcsh:Biology (General) ,A549 Cells ,Drug Design ,biology.protein ,Enzymology ,Parasitology ,lcsh:RC581-607 - Abstract
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., Author Summary Dengue virus (DENV) is the world’s most prevalent mosquito-borne viral disease and nearly 40% of the world’s population is at risk of infection. Currently, no specific drugs are available to treat dengue or other flaviviral diseases. DENV NS5 is a large protein of 900 amino acids composed of two domains with key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of anti-viral inhibitors. We performed a fragment-based screening by X-ray crystallography targeting the DENV NS5 polymerase and identified an allosteric binding pocket at the base of the thumb subdomain close to the enzyme active site. Potent inhibitors active in both DENV polymerase biochemical and cell-based assays were developed through structure-guided design. Resistant virus replicons grown in the presence of the inhibitor, harbored amino acid changes that mapped to the compound binding site. The proposed mode of action for this class of inhibitors is by impeding RdRp protein conformational changes during the transition from initiation to elongation phase of enzyme activity.
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- 2016
32. Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase
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Chin Chin Lim, Yin Hoe Yau, Gang Zou, Pei Yong Shi, Yen Liang Chen, Hongping Dong, Susana Geifman Shochat, Julien Lescar, and Li Jian Yap
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Protein Conformation ,viruses ,Molecular Sequence Data ,RNA-dependent RNA polymerase ,Viral Nonstructural Proteins ,Dengue virus ,Biology ,Virus Replication ,medicine.disease_cause ,Microbiology ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Cricetinae ,RNA polymerase ,Chlorocebus aethiops ,Protein Interaction Mapping ,medicine ,Animals ,Amino Acid Sequence ,Site-directed mutagenesis ,Vero Cells ,Molecular Biology ,Polymerase ,Sequence Homology, Amino Acid ,Flavivirus ,Lysine ,Tryptophan ,RNA ,DNA-Directed RNA Polymerases ,Cell Biology ,RNA-Dependent RNA Polymerase ,Molecular biology ,Enzymes ,chemistry ,Viral replication ,Drug Design ,Mutagenesis, Site-Directed ,biology.protein ,Protein Binding - Abstract
Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.
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- 2011
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33. RNA Structures Required for Production of Subgenomic Flavivirus RNA
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Ezequiel Balmori Melian, Hongping Dong, Alexander A. Khromykh, Judy Edmonds, Nadia Floden, Tomoko Nagasaki, Katherine Truong, Shessy Torres, Pei Yong Shi, and Anneke Funk
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Untranslated region ,Immunology ,Microbiology ,Mice ,Kunjin virus ,Virology ,Exoribonuclease ,Animals ,Guide RNA ,3' Untranslated Regions ,Subgenomic mRNA ,Genetics ,biology ,Flavivirus ,RNA ,biology.organism_classification ,Non-coding RNA ,Genome Replication and Regulation of Viral Gene Expression ,DNA-Binding Proteins ,Insect Science ,Exoribonucleases ,Nucleic Acid Conformation ,RNA, Viral ,Pseudoknot ,West Nile virus - Abstract
Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV KUN ) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.
- Published
- 2010
34. 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members
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Stewart W. Schneller, Michael S. Diamond, Volker Thiel, Jill Schriewer, R. Mark L. Buller, Ganes C. Sen, Theodore C. Pierson, Roland Züst, Soonjeon Youn, Hongping Dong, John S. Errett, Tsai-Yu Lin, Volker Fensterl, Pei Yong Shi, Michael Gale, Kristy J. Szretter, Stephane Daffis, William B. Klimstra, and Jianqing Li
- Subjects
RNA Caps ,Methyltransferase ,viruses ,RNA-binding protein ,Receptor, Interferon alpha-beta ,Biology ,Virus Replication ,Methylation ,Article ,Mice ,03 medical and health sciences ,Interferon ,medicine ,Animals ,Humans ,Gene ,Cells, Cultured ,Adaptor Proteins, Signal Transducing ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Multidisciplinary ,Models, Genetic ,2'-O-methylation ,Poxviridae ,030302 biochemistry & molecular biology ,Models, Immunological ,Proteins ,RNA-Binding Proteins ,RNA ,3T3 Cells ,Methyltransferases ,Fibroblasts ,Virology ,Immunity, Innate ,Neoplasm Proteins ,Coronavirus ,Mice, Inbred C57BL ,Survival Rate ,Gene Expression Regulation ,Protein Biosynthesis ,RNA, Viral ,Interferons ,Apoptosis Regulatory Proteins ,Carrier Proteins ,West Nile virus ,medicine.drug - Abstract
Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.
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- 2010
35. Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase
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Gang Zou, Hongping Dong, Hongmin Li, Yiwei Zhao, Pei Yong Shi, Zhong Li, Lihui Liu, and Siew Pheng Lim
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Models, Molecular ,RNA Caps ,Adenosine ,Antifungal Agents ,RNA capping ,Viral protein ,RNA methylation ,viruses ,Molecular Sequence Data ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Sinefungin ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Binding Sites ,Molecular Structure ,Flavivirus ,virus diseases ,RNA ,Methyltransferases ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Protein Structure, Tertiary ,Viral replication ,RNA, Viral ,West Nile virus - Abstract
The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2'-O positions of the viral RNA cap (GpppA-RNA → m(7)GpppA-RNA → m(7)GpppAm-RNA), using S-adenosyl-l-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-Å resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus, SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.
- Published
- 2010
36. Characterization of Dengue Virus Resistance to Brequinar in Cell Culture
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Zhiming Yuan, Hao Ying Xu, Gang Zou, Hongping Dong, Qing Yin Wang, Min Qing, and Pei Yong Shi
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viruses ,Alphavirus ,Dengue virus ,medicine.disease_cause ,Antiviral Agents ,Virus ,Chlorocebus aethiops ,Drug Resistance, Viral ,medicine ,Animals ,Pharmacology (medical) ,Vero Cells ,Polymerase ,Pharmacology ,biology ,Biphenyl Compounds ,RNA ,Dengue Virus ,biology.organism_classification ,Virology ,Molecular biology ,Flavivirus ,Pyrimidines ,Infectious Diseases ,Vesicular stomatitis virus ,Virion assembly ,biology.protein ,RNA, Viral - Abstract
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.
- Published
- 2010
37. Biochemical and genetic characterization of dengue virus methyltransferase
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David C. Chang, Gang Zou, Siew Pheng Lim, Hongping Dong, Pei Yong Shi, Ka Yan Chung, Julien Lescar, Xuping Xie, and Ying Xiu Toh
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Models, Molecular ,RNA Caps ,Methyltransferase ,viruses ,Mutant ,Antiviral target ,Flavivirus replication ,RNA cap methylation ,Dengue virus ,Biology ,Transfection ,Virus Replication ,medicine.disease_cause ,Methylation ,Cell Line ,Viral Proteins ,Cricetinae ,Virology ,medicine ,Animals ,Replicon ,Polymerase ,Mutation ,virus diseases ,Methyltransferases ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,Viral replication ,biology.protein ,RNA, Viral - Abstract
We report that dengue virus (DENV) methyltransferase sequentially methylates the guanine N-7 and ribose 2'-O positions of viral RNA cap (GpppA-->m(7)GpppA-->m(7)GpppAm). The order of two methylations is determined by the preference of 2'-O methylation for substrate m(7)GpppA-RNA to GpppA-RNA, and the 2'-O methylation is not absolutely dependent on the prior N-7 methylation. A mutation that completely abolished the 2'-O methylation attenuated DENV replication in cell culture, whereas another mutation that abolished both methylations was lethal for viral replication, suggesting that N-7 methylation is more important than 2'-O methylation in viral replication. The latter mutant with lethal replication could be rescued by trans complementation using a wild-type DENV replicon. Furthermore, we found that chimeric DENVs containing the West Nile virus methyltransferase, polymerase, or full-length NS5 were nonreplicative, but the replication defect could also be rescued through trans complementation using the wild-type DENV replicon.
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- 2010
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38. Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel
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Dieter Mueller, Pornwaratt Niyomrattanakit, J. Frasier Glickman, Hongping Dong, Min Qing, Zheng Yin, Thomas H. Keller, Hans Voshol, Kai Lin, Joanne Y.H. Lim, Shahul Nilar, Pei Yong Shi, and Yen Liang Chen
- Subjects
viruses ,Hepatitis C virus ,Immunology ,Drug Evaluation, Preclinical ,Dengue virus ,Biology ,medicine.disease_cause ,Antiviral Agents ,Microbiology ,Primer extension ,Dengue ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,Virology ,RNA polymerase ,medicine ,Animals ,ortho-Aminobenzoates ,Binding site ,Polymerase ,Subgenomic mRNA ,Binding Sites ,virus diseases ,RNA ,Dengue Virus ,RNA-Dependent RNA Polymerase ,Sulfinic Acids ,Molecular biology ,Genome Replication and Regulation of Viral Gene Expression ,chemistry ,Insect Science ,biology.protein ,RNA, Viral ,Allosteric Site - Abstract
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N -sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G) 20 ]. Chemical modification of the initial “hit” improved the compound potency to an IC 50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC 50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC 50 s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.
- Published
- 2010
39. Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants
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Cheng-Feng Qin, Hongping Dong, Xuping Xie, Sumathy Velumani, Pei Yong Shi, Katja Fink, Melissa Hui Yen Chng, Chao Shan, Jassia Pang, Jing Zou, Roland Züst, Evan W. Newell, Shihua Li, and Ying Xiu Toh
- Subjects
Male ,RNA viruses ,0301 basic medicine ,Serotype ,Viral Diseases ,Physiology ,viruses ,Mutant ,lcsh:Medicine ,Monkeys ,Dengue virus ,Pathology and Laboratory Medicine ,medicine.disease_cause ,Biochemistry ,Dengue fever ,Mice ,White Blood Cells ,0302 clinical medicine ,Animal Cells ,Immune Physiology ,Medicine and Health Sciences ,Enzyme-Linked Immunoassays ,lcsh:Science ,Immune Response ,Mammals ,Vaccines ,Mutation ,Immune System Proteins ,Multidisciplinary ,T Cells ,Eukaryota ,virus diseases ,3. Good health ,Infectious Diseases ,Medical Microbiology ,Viral Pathogens ,Viruses ,Vertebrates ,Female ,Pathogens ,Cellular Types ,Research Article ,Primates ,Infectious Disease Control ,Immune Cells ,Immunology ,030231 tropical medicine ,Dengue Vaccines ,Enzyme-Linked Immunosorbent Assay ,Biology ,Research and Analysis Methods ,Microbiology ,Antibodies ,Virus ,Cell Line ,03 medical and health sciences ,medicine ,Animals ,Humans ,Viremia ,Immunoassays ,Microbial Pathogens ,Dengue vaccine ,Blood Cells ,Flaviviruses ,lcsh:R ,Organisms ,Biology and Life Sciences ,Proteins ,Methyltransferases ,Cell Biology ,Dengue Virus ,medicine.disease ,Antibodies, Neutralizing ,Virology ,Macaca fascicularis ,030104 developmental biology ,Cell culture ,Amniotes ,Immunologic Techniques ,lcsh:Q - Abstract
Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.
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- 2018
40. Identification and characterization of inhibitors of West Nile virus
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Hongping Dong, Zhiming Yuan, Pei Yong Shi, Gang Zou, Francesc Puig-Basagoiti, Bo Zhang, and Min Qing
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Transcription, Genetic ,viruses ,Drug Evaluation, Preclinical ,RNA-dependent RNA polymerase ,Biology ,Antiviral Agents ,Article ,Virus ,Inhibitory Concentration 50 ,Viral Proteins ,chemistry.chemical_compound ,Virology ,RNA polymerase ,Chlorocebus aethiops ,Animals ,Flavivirus Infections ,Replicon ,Vero Cells ,Pharmacology ,Viral translation ,virus diseases ,RNA ,Methyltransferases ,RNA-Dependent RNA Polymerase ,biology.organism_classification ,Flavivirus ,chemistry ,Protein Biosynthesis ,RNA, Viral ,West Nile virus - Abstract
Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. To identify flavivirus inhibitors, we performed a high-throughput screening of compound libraries using cells containing luciferase-reporting replicon of West Nile viruses (WNV). Five novel small molecular inhibitors of WNV were identified from libraries containing 96,958 compounds. The inhibitors suppress epidemic strain of WNV in cell culture, with EC(50) (50% effective concentration) values of10microM and TI (therapeutic index) values of10. Viral titer reduction assays, using various flaviviruses and nonflaviviruses, showed that the compounds have distinct antiviral spectra. Mode-of-action analysis showed that the inhibitors block distinct steps of WNV replication: four compounds inhibit viral RNA syntheses, while the other compound suppresses both viral translation and RNA syntheses. Biochemical enzyme assays showed that two compounds selectively inhibit viral RNA-dependent RNA polymerase (RdRp), while another compound specifically inhibits both RdRp and methyltransferase. The identified compounds could potentially be developed for treatment of flavivirus infections.
- Published
- 2009
41. Co-selection of West Nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance RNA/RNA base pairings
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Bo Zhang, David A. Stein, Hongping Dong, and Pei Yong Shi
- Subjects
Models, Molecular ,Base pair ,Molecular Sequence Data ,Genome cyclization ,Adaptation, Biological ,RNA cis elements ,Flavivirus replication ,Viral Plaque Assay ,Antiviral therapy ,Biology ,medicine.disease_cause ,Antiviral Agents ,Article ,Conserved sequence ,03 medical and health sciences ,Virology ,Drug Resistance, Viral ,medicine ,Point Mutation ,Nucleotide ,Selection, Genetic ,Base Pairing ,030304 developmental biology ,Genetics ,chemistry.chemical_classification ,0303 health sciences ,Mutation ,Base Sequence ,030306 microbiology ,Oligonucleotide ,Point mutation ,Mutagenesis ,RNA ,Oligonucleotides, Antisense ,chemistry ,Nucleic Acid Conformation ,West Nile virus - Abstract
West Nile virus (WNV) genome cyclization is mediated by two pairs of long-distance RNA/RNA interactions: the 5′CS/3′CSI (conserved sequence) and the 5′UAR/3′UAR (upstream AUG region) base pairings. Antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), designed to interfere with the 5′CS/3′CSI or 5′UAR/3′UAR base pairings, were previously shown to inhibit WNV. In this study, we selected and characterized WNVs resistant to a PPMO targeting the 3′UAR (3′UAR-PPMO). All resistant viruses accumulated one-nucleotide mutations within the 3′UAR, leading to a single-nucleotide mismatch or a weakened base-pairing interaction with the 3′UAR-PPMO. Remarkably, a one-nucleotide mutation within the 5′UAR was correspondingly co-selected; the 5′UAR mutation restored the base pairing with the 3′UAR mutation. Mutagenesis of WNV demonstrated that the single-nucleotide change within the 3′UAR-PPMO-target site conferred the resistance. RNA binding analysis indicated that the single-nucleotide change reduced the ability of 3′UAR-PPMO to block the RNA/RNA interaction required for genome cyclization. The results suggest a mechanism by which WNV develops resistance to 3′UAR-PPMO, through co-selection of the 5′UAR and 3′UAR, to create a mismatch or a weakened base-pairing interaction with the PPMO, while maintaining the 5′UAR/3′UAR base pairings.
- Published
- 2008
42. West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions
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Bo Zhang, Hongping Dong, Pei Yong Shi, Patrick L. Iversen, and David A. Stein
- Subjects
viruses ,Morpholines ,Genome cyclization ,RNA-dependent RNA polymerase ,RNA cis elements ,Flavivirus replication ,Genome, Viral ,Antiviral therapy ,Biology ,Origin of replication ,Transfection ,Virus Replication ,Cell Line ,Morpholinos ,03 medical and health sciences ,Transcription (biology) ,Cricetinae ,Virology ,Chlorocebus aethiops ,Animals ,3' Untranslated Regions ,Vero Cells ,030304 developmental biology ,Genetics ,0303 health sciences ,030306 microbiology ,Intron ,RNA ,Non-coding RNA ,RNA silencing ,Viral replication ,Mutation ,Nucleic Acid Conformation ,RNA, Viral ,5' Untranslated Regions ,West Nile virus - Abstract
West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5′CS/3′CSI (conserved sequence) interaction, a 5′UAR/3′UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5′UARs capable of forming the 5′/3′ viral RNA interaction were replicative. In contrast, WNV containing a 5′UAR mutation that abolished the 5′/3′ viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5′/3′ RNA interaction. The 5′UAR/3′UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5′UAR/3′UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3′UAR could alternate between pairing with the 5′UAR or with the 3′ end of the flaviviral genome. Therefore, the 5′UAR/3′UAR pairing may release the 3′ end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.
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- 2008
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43. Synergistic Suppression of Dengue Virus Replication Using a Combination of Nucleoside Analogs and Nucleoside Synthesis Inhibitors
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Qing Yin Wang, Yen Liang Chen, Hao Ying Xu, K.L. Yeo, Hongping Dong, Pei Yong Shi, and Fumiaki Yokokawa
- Subjects
Interferon Inducers ,viruses ,Guanosine ,Dengue virus ,Biology ,medicine.disease_cause ,Virus Replication ,Antiviral Agents ,Cell Line ,chemistry.chemical_compound ,Interferon ,Ribavirin ,medicine ,Humans ,Pharmacology (medical) ,Pharmacology ,Interferon inducer ,Nucleoside analogue ,virus diseases ,Drug Synergism ,Nucleosides ,Interferon-beta ,biochemical phenomena, metabolism, and nutrition ,Dengue Virus ,Virology ,Drug Combinations ,Infectious Diseases ,HEK293 Cells ,chemistry ,Pyrimidine metabolism ,Oxidoreductases ,Nucleoside ,medicine.drug - Abstract
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated for antiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs); furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation.
- Published
- 2015
44. Determinants of Dengue Virus NS4A Protein Oligomerization
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Michelle Yueqi Lee, Cheng-Feng Qin, Pei Yong Shi, Hongping Dong, CongBao Kang, Xuping Xie, Jing Zou, Shihua Li, and Chia Min Lee
- Subjects
viruses ,Immunology ,DNA Mutational Analysis ,Dengue virus ,Biology ,Viral Nonstructural Proteins ,medicine.disease_cause ,Virus Replication ,Microbiology ,Virology ,medicine ,Protein oligomerization ,Replicon ,Sequence Deletion ,Genetics ,Structure and Assembly ,Genetic Complementation Test ,Dengue Virus ,biology.organism_classification ,Cell biology ,Complementation ,Transmembrane domain ,Flavivirus ,Viral replication ,Amino Acid Substitution ,Insect Science ,Viral replication complex ,Protein Multimerization - Abstract
Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication. IMPORTANCE We report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by results showing that mutations of flavivirus conserved residues (E50A and G67A located within TMD1) reduced the oligomerization and stability of the NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrated that flavivirus conserved NS4A residues are important for DENV replication. A successful trans complementation of replication-lethal NS4A mutant virus requires wild-type NS4A in the context of the viral replication complex. The wild-type NS4A protein alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcomes in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at the molecular level. The results also suggest that inhibitor blocking of NS4A oligomerization could be explored for antiviral drug discovery.
- Published
- 2015
45. Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening
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Ka Yan Chung, Pei Yong Shi, Hongping Dong, Pornwaratt Niyomrattanakit, David Beer, Siti Nurdiana Abas, Cheah Chen Seh, Kah Fei Wan, Chang Bok Lee, Siew Pheng Lim, Julien Lescar, Shahul Nilar, Chin Chin Lim, and Alex Chao
- Subjects
Methyltransferase ,Transcription, Genetic ,viruses ,RNA-dependent RNA polymerase ,Microbial Sensitivity Tests ,Dengue virus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Antiviral Agents ,chemistry.chemical_compound ,Apoenzymes ,Virology ,RNA polymerase ,Enzyme Stability ,medicine ,Humans ,Nucleotide ,Cysteine ,Enzyme Inhibitors ,Polymerase ,Pharmacology ,chemistry.chemical_classification ,biology ,RNA ,Dengue Virus ,RNA-Dependent RNA Polymerase ,Molecular biology ,Kinetics ,Enzyme ,chemistry ,biology.protein ,RNA, Viral - Abstract
Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1–4 FL NS5. Heavy metal ions such as Zn++ and Co++ as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.
- Published
- 2015
46. Computational analysis of di-peptides correlated with the optimal temperature in G/11 xylanase
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Hongping Dong, Weilan Shao, Hongge Chen, Liangwei Liu, and Suya Wang
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Biochemistry ,Chemistry ,Thermophile ,Xylanase ,Bioengineering ,Computational analysis ,Food science ,Protein engineering ,Applied Microbiology and Biotechnology ,Thermostability ,Mesophile - Abstract
The di-peptides positively correlated with optimal temperature in G/11 xylanase are computed to be: LA, LG, CD, GD, RY, CH; the negatively ones are: DC, YI, YP, and CP. The comparison of the structures of mesophilic and thermophilic xylanase showed that these di-peptides predominantly occur in beta-turns. These results explain the successful improvement of thermostability by introducing arginines into the xylanase surface area and therefore have implications for xylanase engineering.
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- 2006
47. Correction: Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques
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Roland Züst, Hongping Dong, Xiao-Feng Li, David C. Chang, Bo Zhang, Thavamalar Balakrishnan, Ying-Xiu Toh, Tao Jiang, Shi-Hua Li, Yong-Qiang Deng, Brett R. Ellis, Esther M. Ellis, Michael Poidinger, Francesca Zolezzi, Cheng-Feng Qin, Pei-Yong Shi, and Katja Fink
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lcsh:Immunologic diseases. Allergy ,lcsh:Biology (General) ,Virology ,Immunology ,Genetics ,Correction ,Parasitology ,lcsh:RC581-607 ,lcsh:QH301-705.5 ,Molecular Biology ,Microbiology - Published
- 2013
48. Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques
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Esther M. Ellis, Shihua Li, Tao Jiang, Xiaofeng Li, Pei Yong Shi, Michael Poidinger, David C. Chang, Ying Xiu Toh, Roland Züst, Bo Zhang, Brett R. Ellis, Hongping Dong, Yong Qiang Deng, Cheng-Feng Qin, Thavamalar Balakrishnan, Francesca Zolezzi, and Katja Fink
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lcsh:Immunologic diseases. Allergy ,Immunology ,Dengue Vaccines ,Viremia ,Dengue virus ,Vaccines, Attenuated ,medicine.disease_cause ,Microbiology ,Virus ,Dengue fever ,Dengue ,Mice ,Cricetinae ,Virology ,medicine ,Genetics ,Animals ,Humans ,Antibody-dependent enhancement ,Biology ,lcsh:QH301-705.5 ,Molecular Biology ,Dengue vaccine ,Plaque-forming unit ,Aedes ,biology ,Methyltransferases ,Dengue Virus ,medicine.disease ,biology.organism_classification ,Macaca mulatta ,Mice, Mutant Strains ,HEK293 Cells ,Infectious Diseases ,lcsh:Biology (General) ,Interferon Type I ,Mutation ,Medicine ,Clinical Immunology ,Parasitology ,lcsh:RC581-607 ,Research Article - Abstract
Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response., Author Summary The four serotypes of dengue virus cause severe outbreaks globally in tropical countries with thousands of patients requiring hospitalization. The health care and indirect economic cost of dengue in endemic countries is huge. Despite this, no clinically approved vaccine or antiviral treatment is currently available. Dengue transmission could be stopped with a vaccine that provides full protection to all serotypes. Dengue afflicts many developing countries and a vaccine should therefore be cost-effective and should provide protection with ideally a single injection. Here we present a novel dengue vaccine approach that harbours mutation(s) in the 2′-O-methyltransferase (MTase), a viral enzyme that methylates viral RNA as a strategy to escape the host immune response. Non-methylated RNA is recognized as “foreign” and triggers an interferon response in the cell. The MTase mutant virus is immediately recognized by the host's immune response and hardly has a chance to spread in the organism while an immune response is efficiently triggered by the initially infected cells. Mice and monkeys infected with the mutant virus developed an immune response that fully protected them from a challenge with wild-type virus. Furthermore, we show that MTase mutant dengue virus cannot infect Aedes mosquitoes. Collectively, the results suggest 2′-O-MTase mutant dengue virus as a safe, highly immunogenic vaccine approach.
- Published
- 2013
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49. A multidimensional platform for the purification of non-coding RNA species
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Dumnoensun Pruksakorn, I. Ramesh Babu, Chen Gu, Yan Ling Joy Pang, Pei Yong Shi, Chee Sheng Ng, Fabian Hia, Peter R. Preiser, Hongping Dong, Erin G. Prestwich, Megan E. McBee, Yok Hian Chionh, Chia Hua Ho, Dan Su, Sylvie Alonso, Peter C. Dedon, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, McBee, Megan E., Su, Dan, Pang, Yan Ling Joy, Gu, Chen, Prestwich, Erin, Dedon, Peter C., Indrakanti, Ramesh Babu, and School of Biological Sciences
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RNA, Untranslated ,Plasmodium berghei ,Single sample ,Biology ,Genome ,03 medical and health sciences ,RNA, Transfer ,microRNA ,Genetics ,Humans ,Fluorometry ,Epigenetics ,Direct analysis ,Chromatography, High Pressure Liquid ,030304 developmental biology ,Chromatography, Reverse-Phase ,0303 health sciences ,Science::Biological sciences::Genetics [DRNTU] ,030302 biochemistry & molecular biology ,RNA ,Non-coding RNA ,Mycobacterium bovis ,MicroRNAs ,RNA, Bacterial ,RNA, Ribosomal ,Chromatography, Gel ,RNA, Viral ,Methods Online ,Long ncRNA ,RNA, Protozoan - Abstract
A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes., Singapore-MIT Alliance for Research and Technology, National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES002109)
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- 2013
50. Flavivirus RNA cap methyltransferase: structure, function, and inhibition
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Hui Chen, Hongmin Li, Zhong Li, Pei Yong Shi, Hongping Dong, Jing Zhang, Hua Ling, and Lihui Liu
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Methyltransferase ,Ecology ,Guanine ,viruses ,RNA ,virus diseases ,Context (language use) ,Methylation ,Biology ,biology.organism_classification ,Virology ,Molecular biology ,Article ,Flavivirus ,chemistry.chemical_compound ,chemistry ,RNA polymerase ,Genetics ,Binding site ,Ecology, Evolution, Behavior and Systematics ,Biotechnology - Abstract
Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5' terminal cap 1 structure (m(7)GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m(7)GpppA → m(7)GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.
- Published
- 2011
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