scholarly journals Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein

2003 ◽  
Vol 84 (10) ◽  
pp. 2795-2805 ◽  
Author(s):  
Chun-Tang Chiou ◽  
Chih-Chi Andrew Hu ◽  
Pi-Hsin Chen ◽  
Ching-Len Liao ◽  
Yi-Ling Lin ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Assembly ◽  
Susceptibility Gene ◽  
Immunogold Labelling ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Ns3 Protein ◽  
Associated Proteins ◽  
Golgi Network

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B–NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

scholarly journals Conserved amino acids 193–324 of non-structural protein 3 are a dominant source of peptide determinants for CD4+ and CD8+ T cells in a healthy Japanese encephalitis virus-endemic cohort

2004 ◽  
Vol 85 (5) ◽  
pp. 1131-1143 ◽  
Author(s):  
Priti Kumar ◽  
Paramadevanapalli Sulochana ◽  
Gejjehalli Nirmala ◽  
Maganti Haridattatreya ◽  
Vijaya Satchidanandam
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cd8 T Cells ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Parent Protein ◽  
Ns3 Protein ◽  
Conserved Amino Acids

Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4+ as well as CD8+ T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-γ secretion assays revealed that amino acids (aa) 193–324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14·4 kDa stretch to stimulate IFN-γ production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193–324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219–318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein.

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting -1 PRF and the NS3 Protein

2021 ◽  
Author(s):  
Du Yu ◽  
Yundi Zhao ◽  
Junhui Pan ◽  
Xingmiao Yang ◽  
Zhenjie Liang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Replication ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Ns3 Protein

scholarly journals ZBTB38 in Porcine Alveolar Macrophages Positively Regulates the Proliferation of Japanese Encephalitis Virus

2021 ◽  
Author(s):  
Yi Zheng ◽  
Yu-Yong Zhou ◽  
Chun-Xia Chai ◽  
San-Jie Cao ◽  
Qi-Gui Yan ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Alveolar Macrophages ◽  
Encephalitis Virus ◽  
Porcine Alveolar Macrophage ◽  
Envelope Gene ◽  
Interacting Protein ◽  
Btb Domain ◽  
New Information ◽  
Ns3 Protein

Abstract Background Japanese encephalitis (JE) is an important zoonotic disease caused by Japanese encephalitis virus (JEV), and pigs are intermediate host of this disease. Previous studies have confirmed that JEV can proliferate in the respiratory tract of mice and spread through it. Therefore, this study aimed to screen the proteins interacting with JEV on porcine alveolar macrophage cell and verify its role in the proliferation of JEV.Methods and results Porcine alveolar macrophages cell line 3D4/21 were infected with JEV, and obvious cytopathic effect (CPE) was observed. Zinc finger and BTB domain containing 38 (ZBTB38) was screened out as an interacting protein using co-immunoprecipitation assay and validated through knockout and overexpression of ZBTB38 in 3D4/21 cells. The results demonstrated that loss of ZBTB38 function basically had no effect on the attachment and entry processes of JEV, while the transcription level of JEV envelope gene, the expression level of NS3 protein and the number of virions were all significantly down-regulated in the subsequent infection stage. Conclusion Overall, one core conclusion was drawn in this paper that ZBTB38 promotes the proliferation of JEV especially in the middle and late stages of infection. This study provides new information for understanding the pathogenic mechanism of JEV, especially the respiratory transmission caused by JEV infection.

scholarly journals Fusion PCR generated Japanese encephalitis virus/dengue 4 virus chimera exhibits lack of neuroinvasiveness, attenuated neurovirulence, and a dual-flavi immune response in mice

2004 ◽  
Vol 85 (9) ◽  
pp. 2503-2513 ◽  
Author(s):  
Edward Gitau Matumbi Mathenge ◽  
Maria del Carmen Parquet ◽  
Yasutomo Funakoshi ◽  
Seiji Houhara ◽  
Pooi Fong Wong ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Line ◽  
Japanese Encephalitis ◽  
Secondary Infection ◽  
Neuronal Cell ◽  
Protein S ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Rapid Preparation

The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3·17×104 f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100×LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210 000 and 37 000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.

scholarly journals Effect of brefelidin A and monensin on Japanese encephalitis virus maturation and virus release from cells

2011 ◽  
Vol 2 (1) ◽  
pp. 9
Author(s):  
Vaibhavi Jawahar Lad ◽  
Ashok Kumar Gupta
Keyword(s):  
Cell Surface ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Golgi Complex ◽  
Protein Transport ◽  
Virus Assembly ◽  
Brefeldin A ◽  
Antigen Expression ◽  
Encephalitis Virus ◽  
The Individual

Japanese encephalitis virus (JEV) replicates in a variety of cells, the exact intracellular site of virus assembly is somewhat obscure. The aims of this study were to investigate the role Golgi apparatus in JEV maturation by utilizing two Golgi-disrupting agents- brefeldin A (BFA) and monensin (MN) that inhibit virus assembly at specific cellular sites. JEV-infected porcine kidney stable (PS) cells were treated with BFA (2 ug/ mL) or MN (10 uM/ mL) at different h post-infection (p. i.) and the virus contents were assayed after 48 h p. i. The treated cells were further subjected to immuno-fluorescence (IF) using antibodies directed against JEV envelope glycoprotein (gpE) for localization of intracellular viral antigen as well as the antigen expression on the cell surface. Addition of BFA or MN to cells immediately after virus adsorption or at 4 h and 12 h postinfection (p. i.), resulted in 4- or 8- fold reduction in infectious virus contents along with inhibition of its transport to the cell surface, indicating an essential role of the Golgi-associated membranes in JEV replication. Interestingly, the antigenicity of the virus, in contrast, remained unaffected as no difference in epitope presentation/ expression was observed in BFA/MN-treated and control (untreated) infected cells even though in the former cells a loss of hemagglutinating (HA) activity was observed. Further, BFA addition at 18 h or 24 h p. i. showed only a negligible effect on virus suggesting that once the viral-associated membranes are formed, these membranes appear to be stable. In contrast, the inhibition with MN persisted even after its addition to cells at 18 h and 24 h p. i., indicating its sustained effect on JEV. Although BFA inhibits protein transport from endoplasmic reticulum (ER) to the Golgi complex while MN inhibits transport from medial to trans cisternae of the Golgi complex, none of the two agents however affected the gpE synthesis and folding essentially required for the epitope presentation/expression within the cells. As flaviviruses are known to encode three glycoproteins (gps) within their genomes i. e., prM, E, and NS, it will be worthwhile in future to determine whether vesicular transport occurs within or between the virus-induced membranes and how the individual JEV-encoded proteins are transported to discrete compartments further remain to be seen.

scholarly journals Characterizationof the NTPase Activity of Japanese Encephalitis Virus NS3 Protein

1996 ◽  
Vol 77 (9) ◽  
pp. 2077-2084 ◽  
Author(s):  
M.-D. Kuo ◽  
C. Chin ◽  
S.-L. Hsu ◽  
J.-Y. Shiao ◽  
T.-M. Wang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Ns3 Protein

scholarly journals Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus

2019 ◽  
Vol 20 (22) ◽  
pp. 5719
Author(s):  
Yu-Qin Cao ◽  
Lei Yuan ◽  
Qin Zhao ◽  
Jian-Lin Yuan ◽  
Chang Miao ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Viral Entry ◽  
Time Course ◽  
Encephalitis Virus ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Structural Protein ◽  
Viral Binding ◽  
East And Southeast Asia

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

scholarly journals Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1β Expression

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Hui Jiang ◽  
Lige Bai ◽  
Lina Ji ◽  
Zhuofang Bai ◽  
Jianwei Su ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Computational Models ◽  
Encephalitis Virus ◽  
Site Directed Mutagenesis ◽  
Interleukin 1Β ◽  
Mirna Regulation ◽  
Ns3 Protein ◽  
Virus Serotype

ABSTRACT Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation. IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

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