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1.
Mikrochim Acta ; 188(6): 206, 2021 05 27.
Article in English | MEDLINE | ID: mdl-34046739

ABSTRACT

A portable surface-enhanced Raman scattering (SERS)-lateral flow immunoassay (LFIA) detector has been developed for the automatic and highly sensitive detection of West Nile virus (WNV) non-structural protein 1 (NS1) and actual WNV samples. Au@Ag nanoparticles (Au@Ag NPs) labeled with double-layer Raman molecules were used as SERS tags to prepare WNV-specific SERS-LFIA strips. On this platform, the WNV-specific antigen NS1 protein was quantitatively and sensitively detected. The detection limit for the WNV NS1 protein was 0.1 ng/mL, which was 100-fold more sensitive than visual signals. The detection limit for inactivated WNV virions was 0.2 × 102 copies/µL. The sensitivity of the SERS-LFIA detector was comparable to that of the fluorescence quantitative reverse transcription-polymerase chain reaction assay. The prepared SERS-LFIA strips exhibited high sensitivity and good specificity for WNV. Thus, the strips developed herein have clinical application value. Moreover, the portable SERS-LFIA detector enabled automatic and rapid detection of the SERS-LFIA strips. The platform established herein is expected to make a substantial contribution to the diagnosis and control of outbreaks of emerging infectious diseases, including WNV.


Subject(s)
Immunoassay/methods , Spectrum Analysis, Raman/methods , Viral Nonstructural Proteins/analysis , West Nile virus/chemistry , Antibodies, Immobilized/immunology , Gold/chemistry , Limit of Detection , Metal Nanoparticles/chemistry , Silver/chemistry , Viral Nonstructural Proteins/immunology , West Nile Fever/diagnosis
2.
Virology ; 559: 131-144, 2021 07.
Article in English | MEDLINE | ID: mdl-33866234

ABSTRACT

The involvement of the nucleus during flavivirus infection has been observed in only a small number of cases and can be limited to primarily two viral proteins; the structural protein C and the RNA polymerase NS5. Previously we observed that by blocking nuclear transport, WNV strain Kunjin (WNVKUN) replication is severely affected and through mutation of the identified NLS in WNVKUN NS5 protein. In this study, we interrogated the potential nuclear functions of WNVKUN NS5 has on the host transcriptome, by means of RNA sequencing (RNAseq). In a direct comparison between wild type and mutant NS5, it can also be determined that the nuclear translocation of NS5 results in a significant down-regulation of host genes involved in the innate immune response. When compared to published RNAseq data from WNV infection, many of these genes were overlapping indicting the role of NS5 induced transcription during infection.


Subject(s)
Cell Nucleus/virology , Gene Expression , Host Microbial Interactions/genetics , Viral Nonstructural Proteins/metabolism , West Nile virus/chemistry , Down-Regulation , HEK293 Cells , Host Microbial Interactions/immunology , Humans , Immunity, Innate/genetics , Nuclear Localization Signals , Protein Transport , Sequence Analysis, RNA , Up-Regulation , West Nile virus/genetics , West Nile virus/immunology , West Nile virus/metabolism
3.
Methods Mol Biol ; 2266: 227-238, 2021.
Article in English | MEDLINE | ID: mdl-33759130

ABSTRACT

Three-dimensional pharmacophore models have been proven extremely valuable in exploring novel chemical space through virtual screening. However, traditional pharmacophore-based approaches need ligand information and rely on static snapshots of highly dynamic systems. In this chapter, we describe PyRod, a novel tool to generate three-dimensional pharmacophore models based on water traces of a molecular dynamics simulation of an apo-protein.The protocol described herein was successfully applied for the discovery of novel drug-like inhibitors of West Nile virus NS2B-NS3 protease. By using this recent example, we highlight the key steps of the generation and validation of PyRod-derived pharmacophore models and their application for virtual screening.


Subject(s)
Drug Discovery/methods , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Software , Viral Proteases/chemistry , Water/chemistry , Binding Sites , Ligands , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Protein Binding , Small Molecule Libraries , Viral Nonstructural Proteins/chemistry , West Nile virus/chemistry
4.
Anal Chem ; 92(19): 13050-13057, 2020 10 06.
Article in English | MEDLINE | ID: mdl-32854497

ABSTRACT

Absorbance detection is often prohibited in microfluidic channels due to the limited optical path length available in these systems. However, this optical distance may be significantly increased by guiding the probing light beam along the channel length via multiple reflections by patterned metallic surfaces. In this work, we demonstrate enhanced absorbance detection in glass microfluidic channels using a commercial microplate reader based on this principle, yielding detection limits comparable to that measured on standard microwell plates. This improvement in detectability was realized through careful optimization of the mirror lengths and locations combined with the appropriate design of a microchip holder to suitably position the microchannels in the microplate reader. Additionally, it was determined that the angle by which our device was tilted relative to the horizontal plane played an important role in this optimization. For an optimum choice of parameters accessible with our design, the sensitivity of our absorbance measurements in a 30 µm-deep channel was improved by as much as 52-fold, raising this quantity to about 84% of the corresponding value realized for 75 µL samples placed within 7 mm i.d. standard cylindrical microwells. Quantitative ELISAs employing the absorbance detection method were demonstrated on the noted multireflection microchip device for assessing West Nile viral IgM antibody levels in human serum samples yielding analyte detection limits comparable to that measured on standard microwell plates.


Subject(s)
Immunoglobulin M/blood , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , West Nile virus/chemistry , Humans , Microfluidic Analytical Techniques/instrumentation
5.
Biochem Biophys Res Commun ; 531(4): 522-527, 2020 10 22.
Article in English | MEDLINE | ID: mdl-32807496

ABSTRACT

Flaviviruses are major emerging human pathogenic viruses that pose a persistent and growing menace to global health. They are enveloped single-stranded RNA viruses with positive polarity transmitted by arthropod vectors like mosquitoes or ticks, responsible for a significant and growing number of human deaths and illnesses. The 5'- and 3'-untranslated regions (UTRs) are highly structured and contain conserved cis-acting RNA elements that participate in viral translation, replication and host adaptation. Despite their role in fiaviviruses replication, few high-resolution structural studies have investigated the RNA elements required for viral replication. Here we report the NMR structures of stem-loop B from WNV and DENV4 viruses. Because this element is required for cyclization of the genome and the activity of the replicative viral enzymes, viral replication rates are sensitive to even small changes in these RNAs. Therefore, this work provides structural insight into a new drug target to reduce flavivirus replication rates.


Subject(s)
Dengue Virus/chemistry , RNA, Viral/chemistry , West Nile virus/chemistry , 5' Untranslated Regions , Dengue Virus/physiology , Magnetic Resonance Spectroscopy , Models, Molecular , Mutation , Nucleic Acid Conformation , Temperature , Virus Replication
6.
Front Immunol ; 11: 16, 2020.
Article in English | MEDLINE | ID: mdl-32038660

ABSTRACT

West Nile (WN) virus infection of humans is frequently asymptomatic, but can also lead to WN fever or neuroinvasive disease. CD4 T cells and B cells are critical in the defense against WN virus, and neutralizing antibodies, which are directed against the viral glycoprotein E, are an accepted correlate of protection. For the efficient production of these antibodies, B cells interact directly with CD4 helper T cells that recognize peptides from E or the two other structural proteins (capsid-C and membrane-prM/M) of the virus. However, the specific protein sites yielding such helper epitopes remain unknown. Here, we explored the CD4 T cell response in humans after WN virus infection using a comprehensive library of overlapping peptides covering all three structural proteins. By measuring T cell responses in 29 individuals with either WN virus disease or asymptomatic infection, we showed that CD4 T cells focus on peptides in specific structural elements of C and at the exposed surface of the pre- and postfusion forms of the E protein. Our data indicate that these immunodominant epitopes are recognized in the context of multiple different HLA molecules. Furthermore, we observed that immunodominant antigen regions are structurally conserved and similarly targeted in other mosquito-borne flaviviruses, including dengue, yellow fever, and Zika viruses. Together, these findings indicate a strong impact of virion protein structure on epitope selection and antigenicity, which is an important issue to consider in future vaccine design.


Subject(s)
Asymptomatic Infections , CD4-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , West Nile Fever/immunology , West Nile virus/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Capsid Proteins/immunology , Cohort Studies , Dengue Virus/chemistry , Dengue Virus/immunology , Epitopes, T-Lymphocyte/chemistry , Female , HLA-D Antigens/genetics , Humans , Immunodominant Epitopes/immunology , Male , Middle Aged , Peptide Library , RNA, Viral/blood , Viral Envelope Proteins/immunology , West Nile Fever/virology , West Nile virus/chemistry , Yellow fever virus/chemistry , Yellow fever virus/immunology , Zika Virus/chemistry , Zika Virus/immunology
7.
Antiviral Res ; 175: 104731, 2020 03.
Article in English | MEDLINE | ID: mdl-32014497

ABSTRACT

West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts.


Subject(s)
Dengue Virus/enzymology , Models, Molecular , Viral Nonstructural Proteins/metabolism , West Nile virus/enzymology , Zika Virus/enzymology , Binding Sites , Combinatorial Chemistry Techniques , Dengue Virus/chemistry , Drug Development , Kinetics , RNA Helicases/chemistry , RNA Helicases/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Structure-Activity Relationship , Substrate Specificity , Viral Nonstructural Proteins/chemistry , West Nile virus/chemistry , Zika Virus/chemistry
8.
Sci Rep ; 10(1): 1163, 2020 01 24.
Article in English | MEDLINE | ID: mdl-31980725

ABSTRACT

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.


Subject(s)
Binding Sites , Ligands , Membrane Proteins/metabolism , Proteomics/methods , Receptors, Cell Surface/metabolism , Adhesins, Bacterial/chemistry , Adhesins, Bacterial/metabolism , Amino Acids , Collodion , Endothelial Cells/metabolism , Immobilized Proteins , Membrane Proteins/chemistry , Membranes, Artificial , Neisseria meningitidis/chemistry , Polyvinyls , Protein Binding , Protein Domains , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , West Nile virus/chemistry
9.
J Med Chem ; 63(1): 140-156, 2020 01 09.
Article in English | MEDLINE | ID: mdl-31804823

ABSTRACT

The ß-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with ß-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-ß-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography-mass spectrometry experiments revealed that tripeptide-ß-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the ß-lactam ring with the catalytic serine, followed by release of the inhibitor with opened ß-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.


Subject(s)
Antiviral Agents/pharmacology , Oligopeptides/pharmacology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , beta-Lactams/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Catalytic Domain , Cell Line, Tumor , Dengue Virus/chemistry , Dengue Virus/drug effects , Dipeptides/chemical synthesis , Dipeptides/metabolism , Dipeptides/pharmacology , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Protein Binding , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/metabolism , Viral Nonstructural Proteins/metabolism , West Nile virus/chemistry , West Nile virus/drug effects , beta-Lactams/chemical synthesis , beta-Lactams/metabolism
10.
Protein Expr Purif ; 153: 18-25, 2019 01.
Article in English | MEDLINE | ID: mdl-30125621

ABSTRACT

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.


Subject(s)
Antibodies, Viral/biosynthesis , Antigens, Viral/immunology , Encephalitis Virus, St. Louis/immunology , Encephalitis, St. Louis/diagnosis , Viral Nonstructural Proteins/immunology , West Nile Fever/diagnosis , West Nile virus/immunology , Animals , Antibody Specificity , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Argentina , Blotting, Western , Chromatography, Affinity , Cloning, Molecular , Cross Reactions , Diagnosis, Differential , Encephalitis Virus, St. Louis/chemistry , Encephalitis Virus, St. Louis/genetics , Encephalitis, St. Louis/immunology , Encephalitis, St. Louis/virology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Inclusion Bodies/chemistry , Mice , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Solubility , Viral Nonstructural Proteins/biosynthesis , Viral Nonstructural Proteins/genetics , West Nile Fever/immunology , West Nile Fever/virology , West Nile virus/chemistry , West Nile virus/genetics
11.
Anal Chem ; 90(12): 7777-7783, 2018 06 19.
Article in English | MEDLINE | ID: mdl-29790331

ABSTRACT

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 µL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.


Subject(s)
Electrochemical Techniques , Paper , Virion/chemistry , Virion/isolation & purification , West Nile virus/chemistry , West Nile virus/isolation & purification , Gold/chemistry , Molecular Structure
12.
Nat Struct Mol Biol ; 25(1): 13-20, 2018 01.
Article in English | MEDLINE | ID: mdl-29323278

ABSTRACT

Zika virus (ZIKV) is an enveloped, icosahedral flavivirus that has structural and functional similarities to other human flavivirus pathogens such as dengue (DENV), West Nile (WNV) and Japanese encephalitis (JEV) viruses. ZIKV infections have been linked to fetal microcephaly and the paralytic Guillain-Barré syndrome. This review provides a comparative structural analysis of the assembly, maturation and host-cell entry of ZIKV with other flaviviruses, especially DENV. We also discuss the mechanisms of neutralization by antibodies.


Subject(s)
Virus Assembly , Virus Internalization , Zika Virus Infection/virology , Zika Virus/chemistry , Zika Virus/physiology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cryoelectron Microscopy , Dengue Virus/chemistry , Dengue Virus/physiology , Encephalitis Virus, Japanese/chemistry , Encephalitis Virus, Japanese/physiology , Female , Guillain-Barre Syndrome/virology , Humans , Male , Mice , Microcephaly/virology , Models, Biological , Pregnancy , Protein Conformation , United States , West Nile virus/chemistry , West Nile virus/physiology
13.
Antiviral Res ; 146: 174-183, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28927677

ABSTRACT

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC50 ∼ 500 µM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3pro. To identify the matching protein binding site, chemical shift perturbation studies employing 1H,15N-TROSY-HSQC experiments with uniformly 2H,15N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3pro inhibitors.


Subject(s)
Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects , West Nile virus/drug effects , Binding Sites , Drug Design , Humans , Magnetic Resonance Spectroscopy , Protein Conformation , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , West Nile virus/chemistry , West Nile virus/enzymology
14.
Anal Chem ; 89(1): 862-870, 2017 01 03.
Article in English | MEDLINE | ID: mdl-27977168

ABSTRACT

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein-RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.


Subject(s)
Hydrodynamics , RNA, Viral/analysis , T-Cell Intracellular Antigen-1/analysis , Ultracentrifugation , West Nile virus/chemistry , Humans
15.
Virology ; 500: 122-129, 2017 01.
Article in English | MEDLINE | ID: mdl-27816638

ABSTRACT

For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (Yamshchikov et al., 2016). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccine candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue.


Subject(s)
Mutation, Missense , Vaccines, DNA/genetics , Viral Envelope Proteins/genetics , Viral Vaccines/genetics , West Nile Fever/virology , West Nile virus/genetics , Amino Acid Sequence , Animals , Cell Line , Female , Humans , Mice, Inbred ICR , Molecular Sequence Data , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , West Nile Fever/immunology , West Nile Fever/prevention & control , West Nile virus/chemistry , West Nile virus/immunology
16.
Biosens Bioelectron ; 87: 646-653, 2017 Jan 15.
Article in English | MEDLINE | ID: mdl-27619528

ABSTRACT

This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the effect of contaminants in commercial thiolated DNA probe, the electrode surface was functionalized with the 24-nucleotide DNA probes based on the West Nile virus sequence (Kunjin strain). The biosensor has the ability to detect complementary DNA fragments with a detection limit down to 20 DNA target molecules (1.5aM range), making it suitable for a practical point-of-care (POC) platform for low target count clinical applications without the need for amplification. The reproducibility of the biosensor detection was improved with efficient covalent immobilization of purified single-stranded DNA probe oligomers on cleaned gold microelectrodes. In addition to the low detection limit, the biosensor showed a dynamic range of detection from 1µL-1 to 105µL-1 target molecules (20 to 2 million targets), making it suitable for sample analysis in a typical clinical application environment. The binding results presented in this paper were validated using fluorescent oligomers.


Subject(s)
DNA Probes/chemistry , DNA, Single-Stranded/chemistry , DNA/analysis , Electric Capacitance , Electrochemical Techniques/instrumentation , Immobilized Nucleic Acids/chemistry , Nucleic Acid Hybridization , Base Sequence , Biosensing Techniques/instrumentation , Electrodes , Equipment Design , Gold , Humans , Limit of Detection , Point-of-Care Systems , Reproducibility of Results , Sulfhydryl Compounds/chemistry , West Nile Fever/virology , West Nile virus/chemistry
17.
Vaccine ; 34(46): 5479-5482, 2016 11 04.
Article in English | MEDLINE | ID: mdl-27670075

ABSTRACT

A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable.


Subject(s)
Adjuvants, Immunologic , Antibodies, Viral/immunology , Hydrogels , Peptides/immunology , West Nile Fever/prevention & control , West Nile Virus Vaccines/immunology , West Nile virus/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Animals , Immunity, Cellular , Immunity, Humoral , Mice , Protein Domains/immunology , Th1 Cells/immunology , Vaccines, Subunit/immunology , Viral Envelope Proteins/immunology , West Nile Fever/immunology , West Nile Virus Vaccines/administration & dosage , West Nile Virus Vaccines/chemistry , West Nile virus/chemistry
18.
Nature ; 533(7603): 425-8, 2016 05 19.
Article in English | MEDLINE | ID: mdl-27093288

ABSTRACT

Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses, and with Guillian-Barré syndrome in adults. Here we present the 3.7 Å resolution cryo-electron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV. This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryo-electron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen, saliva and urine. Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.


Subject(s)
Temperature , Virion/chemistry , Virion/ultrastructure , Zika Virus/chemistry , Zika Virus/ultrastructure , Cryoelectron Microscopy , Dengue Virus/chemistry , Dengue Virus/classification , Dengue Virus/pathogenicity , Encephalitis Virus, Japanese/chemistry , Humans , Models, Molecular , Protein Stability , Saliva/virology , Semen/virology , Urine/virology , Viral Envelope Proteins/chemistry , Virion/pathogenicity , West Nile virus/chemistry , Zika Virus/pathogenicity
19.
Virology ; 481: 95-106, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25771497

ABSTRACT

The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication.


Subject(s)
Cell Membrane/virology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication , West Nile Fever/virology , West Nile virus/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Conserved Sequence , Humans , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Viral Nonstructural Proteins/genetics , West Nile virus/chemistry , West Nile virus/genetics , West Nile virus/growth & development
20.
Protein Sci ; 24(1): 117-28, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25352331

ABSTRACT

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.


Subject(s)
Dengue Virus/enzymology , Dengue/virology , Methyltransferases/metabolism , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/metabolism , West Nile Fever/virology , West Nile virus/enzymology , Crystallography, X-Ray , Dengue Virus/chemistry , Dengue Virus/metabolism , Humans , Methyltransferases/chemistry , Methyltransferases/isolation & purification , Models, Molecular , Protein Refolding , S-Adenosylhomocysteine/isolation & purification , S-Adenosylmethionine/isolation & purification , West Nile virus/chemistry , West Nile virus/metabolism
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