scholarly journals NS1′ of Flaviviruses in the Japanese Encephalitis Virus Serogroup Is a Product of Ribosomal Frameshifting and Plays a Role in Viral Neuroinvasiveness

2009 ◽  
Vol 84 (3) ◽  
pp. 1641-1647 ◽  
Author(s):  
Ezequiel Balmori Melian ◽  
Edward Hinzman ◽  
Tomoko Nagasaki ◽  
Andrew E. Firth ◽  
Norma M. Wills ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Sequence Elements ◽  
Rna Pseudoknot ◽  
Frameshift Event

ABSTRACT Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1′, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1′ is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1′ is the product of a −1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1′ plays a role in viral neuroinvasiveness.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts

2021 ◽  
Vol 102 (9) ◽  
Author(s):  
Kiran Bala Sharma ◽  
Simran Chhabra ◽  
Suruchi Aggarwal ◽  
Aarti Tripathi ◽  
Arup Banerjee ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Protein Composition ◽  
Enrichment Analysis ◽  
Mouse Embryonic Fibroblast ◽  
Encephalitis Virus ◽  
Innate Immune ◽  
Pathway Enrichment Analysis ◽  
Interferon Stimulated Genes ◽  
Immune Sensing

Advances in proteomics have enabled a comprehensive understanding of host–pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS–STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird’s-eye view into how fibroblast protein composition is rewired following JEV infection.

scholarly journals Histidine at Residue 99 and the Transmembrane Region of the Precursor Membrane prM Protein Are Important for the prM-E Heterodimeric Complex Formation of Japanese Encephalitis Virus

2005 ◽  
Vol 79 (13) ◽  
pp. 8535-8544 ◽  
Author(s):  
Ying-Ju Lin ◽  
Suh-Chin Wu
Keyword(s):  
Complex Formation ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Microscopic Analysis ◽  
Subcellular Location ◽  
Encephalitis Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Heterodimeric Complex ◽  
Prm Protein

ABSTRACT The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.

scholarly journals Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

2014 ◽  
Vol 53 (2) ◽  
pp. 557-566 ◽  
Author(s):  
Day-Yu Chao ◽  
Jedhan Ucat Galula ◽  
Wen-Fan Shen ◽  
Brent S. Davis ◽  
Gwong-Jen J. Chang
Keyword(s):  
West Nile Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Igg Antibody ◽  
West Nile ◽  
Dengue Viruses ◽  
Nonstructural Protein 1 ◽  
Antibody Capture

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.

scholarly journals Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

2007 ◽  
Vol 15 (1) ◽  
pp. 88-94 ◽  
Author(s):  
Eiji Konishi ◽  
Yoko Kitai ◽  
Takashi Kondo
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Test Method ◽  
Assay Method ◽  
Specific Antibodies ◽  
Nonstructural Protein 1 ◽  
Complement Dependent Cytotoxicity ◽  
Low Levels

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

scholarly journals Mutation analysis of the cross-reactive epitopes of Japanese encephalitis virus envelope glycoprotein

2012 ◽  
Vol 93 (6) ◽  
pp. 1185-1192 ◽  
Author(s):  
Shyan-Song Chiou ◽  
Yi-Chin Fan ◽  
Wayne D. Crill ◽  
Ruey-Yi Chang ◽  
Gwong-Jen J. Chang
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Recombinant Expression ◽  
Encephalitis Virus ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Virus Envelope ◽  
E Protein ◽  
Domain Iii

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.

scholarly journals The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

2015 ◽  
Vol 90 (3) ◽  
pp. 1178-1189 ◽  
Author(s):  
Li-Chen Yen ◽  
Jia-Teh Liao ◽  
Hwei-Jen Lee ◽  
Wei-Yuan Chou ◽  
Chun-Wei Chen ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Replication ◽  
Japanese Encephalitis ◽  
Protective Immunity ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
The Core ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
Infected Cells

ABSTRACTNS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using atrans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain.IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

scholarly journals Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

2014 ◽  
Vol 95 (4) ◽  
pp. 806-815 ◽  
Author(s):  
Xiao-Dan Li ◽  
Xiao-Feng Li ◽  
Han-Qing Ye ◽  
Cheng-Lin Deng ◽  
Qing Ye ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cdna Clone ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Full Length ◽  
Large Plaque ◽  
Adaptive Mutations ◽  
A Genome

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

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