scholarly journals Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

2015 ◽  
Vol 11 (3) ◽  
pp. e1004750 ◽  
Author(s):  
Yu-Ting Kao ◽  
Bi-Lan Chang ◽  
Jian-Jong Liang ◽  
Hang-Jen Tsai ◽  
Yi-Ling Lee ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Mitochondrial Trifunctional Protein

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.

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 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 NS5 from Dengue Virus Serotype 2 Can Adopt a Conformation Analogous to That of Its Zika Virus and Japanese Encephalitis Virus Homologues

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Abbas El Sahili ◽  
Tingjin Sherryl Soh ◽  
Jonas Schiltz ◽  
Aïcha Gharbi-Ayachi ◽  
Cheah Chen Seh ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Vaccine Design ◽  
Encephalitis Virus ◽  
Virus Serotype ◽  
In Cells

ABSTRACT Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design. IMPORTANCE Flaviviruses include important human pathogens, such as dengue virus and Zika virus. NS5 is a nonstructural protein essential for flavivirus RNA replication with dual MTase and RdRp enzyme activities and thus constitutes a major drug target. Insights into NS5 structure, dynamics, and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling that of NS5 from JEV and ZIKV. Replacement of the DENV2 NS5 linker with the JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic flavivirus NS5 linkers impede DENV2 NS5 protein-protein interactions that are essential for virus replication.

scholarly journals Antiapoptotic but Not Antiviral Function of Humanbcl-2 Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells

1998 ◽  
Vol 72 (12) ◽  
pp. 9844-9854 ◽  
Author(s):  
Ching-Len Liao ◽  
Yi-Ling Lin ◽  
Shih-Cheng Shen ◽  
Jing-Yih Shen ◽  
Hong-Lin Su ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Lines ◽  
Japanese Encephalitis ◽  
Cho Cells ◽  
Nonstructural Protein ◽  
Chinese Hamster ◽  
Encephalitis Virus ◽  
Stable Expression ◽  
Stable Cell Lines ◽  
Nonstructural Protein 1

ABSTRACT Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of humanbcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog,bcl-XL , following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforcedbcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellularbcl-2 which plays a role in the establishment of JEV persistence.

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