Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

ABSTRACT Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or α-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducibleEscherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or β-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.

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Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.00340-16 ◽

2016 ◽

Vol 90 (12) ◽

pp. 5735-5749 ◽

Cited By ~ 27

Author(s):

Xiao-Dan Li ◽

Cheng-Lin Deng ◽

Han-Qing Ye ◽

Hong-Lei Zhang ◽

Qiu-Yan Zhang ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Protease Activity ◽

Rna Synthesis ◽

Rna Replication ◽

Viral Assembly ◽

Transmembrane Domains ◽

Viral Rna ◽

Virion Assembly ◽

Ns3 Protease

ABSTRACTFlavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly.IMPORTANCEMany flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.

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Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus

Molecules ◽

10.3390/molecules25051246 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1246

Author(s):

Chit Care ◽

Wannapa Sornjai ◽

Janejira Jaratsittisin ◽

Atitaya Hitakarun ◽

Nitwara Wikan ◽

...

Keyword(s):

Dengue Virus ◽

Protein Expression ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Denv Infection ◽

Significant Inhibition ◽

Encephalitis Virus ◽

Host Protein ◽

Cell Protein ◽

Infected Cells

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

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Blocking of the Alpha Interferon-Induced Jak-Stat Signaling Pathway by Japanese Encephalitis Virus Infection

Journal of Virology ◽

10.1128/jvi.78.17.9285-9294.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9285-9294 ◽

Cited By ~ 136

Author(s):

Ren-Jye Lin ◽

Ching-Len Liao ◽

Elong Lin ◽

Yi-Ling Lin

Keyword(s):

Japanese Encephalitis Virus ◽

Signaling Pathway ◽

Tyrosine Phosphorylation ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Janus Kinase ◽

Luciferase Reporter ◽

Dependent Manner ◽

Stat Signaling ◽

Infected Cells

ABSTRACT The induction of alpha/beta interferon (IFN-α/β) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-α had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-α in the cultured cell system. JEV infection appeared to render cells resistant to IFN-α since the IFN-α-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-α/β receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-α stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-α/β by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.

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

Journal of Virology ◽

10.1128/jvi.02057-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1178-1189 ◽

Cited By ~ 2

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.

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Differential miRNA Expression Profiling Reveals Correlation of miR125b-5p with Persistent Infection of Japanese Encephalitis Virus

International Journal of Molecular Sciences ◽

10.3390/ijms22084218 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4218

Author(s):

Chih-Wei Huang ◽

Kuen-Nan Tsai ◽

Yi-Shiuan Chen ◽

Ruey-Yi Chang

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Persistent Infection ◽

Quantitative Polymerase Chain Reaction ◽

Acute Infection ◽

Encephalitis Virus ◽

Luciferase Reporter ◽

Northern Analysis ◽

Genome Replication ◽

Infected Cells

MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA’s involvement in flavivirus persistent infection. Here, we used an miRNA array analysis of Japanese encephalitis virus (JEV)-infected cells to search for persistent infection-associated miRNAs in comparison to acute infection. Among all differentially expressed miRNAs, the miR-125b-5p is the most significantly increased one. The high level of miR-125b-5p in persistently JEV-infected cells was confirmed by Northern analysis and real-time quantitative polymerase chain reaction. As soon as the cells established a persistent infection, a significantly high expression of miR-125b-5p was readily observed. Transfecting excess quantities of a miR-125b-5p mimic into acutely infected cells reduced genome replication and virus titers. Host targets of miR125b-5p were analyzed by target prediction algorithms, and six candidates were confirmed by a dual-luciferase reporter assay. These genes were upregulated in the acutely infected cells and sharply declined in the persistently infected cells. The transfection of the miR125b-5p mimic reduced the expression levels of Stat3, Map2k7, and Triap1. Our studies indicated that miR-125b-5p targets both viral and host sequences, suggesting its role in coordinating viral replication and host antiviral responses. This is the first report to characterize the potential roles of miR-125b-5p in persistent JEV infections.

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