Japanese Encephalitis Virus Nonstructural Protein Research Articles

SLC25A12 inhibits Japanese encephalitis virus replication by interacting with the NS1 and enhancing the type I interferon response

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-β expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.

JEV infection leads to dysfunction of lysosome by downregulating the expression of LAMP1 and LAMP2

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What’s more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131–204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Linear epitope identification of monoclonal antibodies against the duck Tembusu virus NS1

Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.

Swine IFI6 confers antiviral effects against Japanese encephalitis virus in vitro and in vivo.

Swine are considered to be an important intermediate host in the cycle of Japanese encephalitis virus (JEV) infection. Most existing antiviral studies of JEV mainly focus on the host factor of the dead-end hosts. However, little research has addressed this in swine. Here, we found that swine interferon alpha-inducible protein 6 (sIFI6) possessed antiviral activity against JEV. In vitro studies showed that overexpression of sIFI6 inhibited the infection of JEV, while sIFI6 knockdown enhanced the infection of JEV in PK-15 cells. In addition, we also found that the structural integrity of sIFI6 was required by anti-JEV activity and that sIFI6 interacted with JEV nonstructural protein 4A (NS4A), an integral membrane protein with a pivotal function in replication complex during JEV replication. The interaction domain was mapped to the fourth transmembrane domain (TMD), also known as the 2K peptide of NS4A. The antiviral activity of sIFI6 was regulated by endoplasmic reticulum (ER) stress-related protein, Bip. In vivo studies revealed that sIFI6 alleviated symptoms of JEV infection in C57BL/6 mice. In addition, the antiviral spectrum of sIFI6 showed that sIFI6 specifically inhibited JEV infection. In conclusion, this study identified sIFI6 as a host factor against JEV infection for the first time. Our findings provide a potential drug target against JEV infection.

Proteomic analyses identify intracellular targets for Japanese encephalitis virus nonstructural protein 1 (NS1)

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus (JEV). JEV nonstructural protein 1 (NS1) is involved in many crucial biological events during viral infection and immune suppression. To investigate the role of JEV NS1 in virus-infected cells, the molecules with which it interacts intracellularly were screened with a pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The interaction between heterogeneous nuclear ribonucleoprotein K (hnRNP K), vimentin and NS1 were verified with coimmunoprecipitation and confocal assays. Our results show that JEV NS1 interacts with vimentin, hnRNP K and colocalizes with cellular vimentin and hnRNP K. Furthermore, our results demonstrate that the expression of vimentin and hnRNP K were up-regulated in both NS1-transfected and JEV-infected cells. Knocking down vimentin and hnRNP K reduced viral replication while conversely, over-expression of vimentin and hnRNP K improved viral replication, suggesting an important role for this protein in the viral life cycle. Also, We found that vimentin also interacted with hnRNP K after overexpression of NS1 or JEV infection. These findings provide insight into the molecular mechanism of JEV replication and highlight the key role the NS1 in JEV infection.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Identification of a protective epitope in Japanese encephalitis virus NS1 protein

Japanese encephalitis virus (JEV) is one of the most important culex transmitted-flaviviruses, which can cause encephalitis in humans. Although non-structural protein 1 (NS1) of JEV does not stimulate neutralizing antibodies, this protein can provide high immunoprotection in vivo. The protective epitopes and the protective mechanism of NS1 still remain unclear. In this study, we generated five different monoclonal antibodies (mAbs) targeting the NS1 protein of JEV. In vitro experiments revealed that none of these five antibodies neutralized the JEV infection. In mouse protection studies, one of these mAbs, designated 2B8, provided a therapeutic effect against JEV lethal challenge (70% survival rate). Using peptide mapping analysis, we found that mAb 2B8 reacted with the epitope 225PETHTLWGD233 in the NS1 protein, in which any mutations among amino acid residues T228, H229, L231 or W232 could cause binding failure of 2B8 to the NS1 protein. Furthermore, mice immunized with KLH-polypeptide (225PETHTLWGD233) showed reduced mortality following JEV challenge. Collectively, we found a new protective epitope in the JEV NS1 protein. These results may facilitate the development of therapeutic agent and subunit-based vaccines based on the NS1 protein.

Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and immunogenicity

Japanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attempts to express JEV NS1 in E. coli are on due to advantages such as rapid growth, easy manipulation, low cost, and high yield. However, the challenges of low yield and poor solubility of the proteins expressed in E. coli remain to be overcome. Herein, we reported successful expression of the JEV NS1 protein in E. coli Rosetta(DE3) strain. We standardized the temperature, induction time, as well as the concentration of the inducer for optimizing the expression of JEV NS1 in E. coli. Further, we successfully obtained soluble JEV NS1 from inclusion bodies by partial refolding during elution and gradual refolding during dialysis. Furthermore, the JEV NS1 protein was found to retain its molecular weight and was able to induce an immune response in the mouse. Western blot and indirect enzyme-linked immunosorbent assay were performed using the blood of the immunized mouse and purified JEV NS1 in this study. Hence, JEV NS1 expressed in and isolated from E. coli Rosetta(DE3) strain holds potential for application in vaccine development and diagnostic studies to combat Japanese encephalitis outbreaks in the future.

Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection.

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease.

TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A.

The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A.

Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs fatty acid β-oxidation.

Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.

Protection against Dengue Virus Infection in Mice by Administration of Antibodies against Modified Nonstructural Protein 1

BackgroundInfection with dengue virus (DENV) may cause life-threatening disease with thrombocytopenia and vascular leakage which are related to dysfunction of platelets and endothelial cells. We previously showed that antibodies (Abs) against DENV nonstructural protein 1 (NS1) cross-react with human platelets and endothelial cells, leading to functional disturbances. Based on sequence homology analysis, the C-terminal region of DENV NS1 protein contains cross-reactive epitopes. For safety in vaccine development, the cross-reactive epitopes of DENV NS1 protein should be deleted or modified.Methodology/Principal FindingsWe tested the protective effects of Abs against full-length DENV NS1, NS1 lacking the C-terminal amino acids (a.a.) 271-352 (designated ΔC NS1), and chimeric DJ NS1 consisting of N-terminal DENV NS1 (a.a. 1-270) and C-terminal Japanese encephalitis virus NS1 (a.a. 271-352). The anti-ΔC NS1 and anti-DJ NS1 Abs showed a lower binding activity to endothelial cells and platelets than that of anti-DENV NS1 Abs. Passive immunization with anti-ΔC NS1 and anti-DJ NS1 Abs reduced DENV-induced prolonged mouse tail bleeding time. Treatment with anti-DENV NS1, anti-ΔC NS1 and anti-DJ NS1 Abs reduced local skin hemorrhage, controlled the viral load of DENV infection in vivo, synergized with complement to inhibit viral replication in vitro, as well as abolished DENV-induced macrophage infiltration to the site of skin inoculation. Moreover, active immunization with modified NS1 protein, but not with unmodified DENV NS1 protein, reduced DENV-induced prolonged bleeding time, local skin hemorrhage, and viral load.Conclusions/SignificanceThese results support the idea that modified NS1 proteins may represent an improved strategy for safe and effective vaccine development against DENV infection.

Comprehensive mapping of a novel NS1 epitope conserved in flaviviruses within the Japanese encephalitis virus serocomplex

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues 227ETHTLW232. Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

NS1′ protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1' protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a -1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1' protein through the production of JEV NS1'-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1' protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1' protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1' enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues 5AIDITRK11, 72RDELNVL78, 251KSKHNRREGY260, 269DENGIVLD276, and 341DETTLVRS348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

P036 Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs long-chain fatty acid metabolism

Introduction Japanese encephalitis virus (JEV) nonstructural protein NS5 has two enzymatic activities, methyltransferase and RNA-dependent RNA polymerase, and has been identified as a strong antagonist of type I interferon signaling. How NS5 executes these diverse functions is largely unknown. Methods We used immunoprecipitation assay to investigate the cellular proteins interacting with JEV NS5. The interaction of NS5 with cellular protein was confirmed by immunoprecipitation-western assay with NS5 expressed by plasmid transfection and by JEV infection. The defect of long-chain fatty acid metabolism was analyzed through measurement of the oxygen consumption rate (OCR) by XF Extracellular Flux Analyzer. Results Two mitochondrial proteins, hydroxyacyl-CoA dehydrogenase α and β subunits (HADH α and HADH β ) that form mitochondrial trifunctional protein and are involved in long-chain fatty acid β -oxidation, were immunoprecipitated with NS5. Besides the well-known cytoplasmic location, we find that NS5 could also be detected in mitochondria. The deletion constructs of NS5 suggest that the N-terminal methyltransferase domain of NS5 is essential for the interaction with HADH α . Since HADH α and HADH β are involved in long-chain fatty acid metabolism, we measured OCR in cells supplemented with or without long-chain fatty acid palmitate. An enhanced OCR was noted in JEV-infected cells cultured in normal medium, however when palmitate was the only energy source, the OCR was greatly reduced in JEV-infected cells. Conclusion These results suggest that JEV infection requires high energy supply, but long-chain fatty acid β -oxidation is impaired in JEV-infected cells. Impaired mitochondria β -oxidation has been implicated in reactive oxygen species production, cytokine induction, and virus-associated encephalitis, thus the role of NS5 interacting with HADH α /HADH β in blocking long-chain fatty acid metabolism as well as the various functions of NS5 warrants further study.

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1'), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1' could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1' coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.

Japanese encephalitis virus nonstructural protein NS3 has RNA binding and ATPase activities.

Sequence data suggest that Japanese encephalitis virus (JEV) protein NS3 is a multifunctional protein with sequence motifs characteristic of a protease and a helicase. To examine the functions of JEV-NS3, a fusion protein of NS3 in Escherichia coli was generated. Analysis by Western blot using monospecific rabbit antisera generated against the fusion protein (anti-MBJEN3) showed that NS3 was localized in the membrane fraction of JEV-infected cells and the particulate fraction of bacteria extracts. The addition of anti-MBJEN3 sera reduced JEV-specific RNA synthesis activity in a in vitro system. In addition, NS3 was shown to exhibit RNA binding and ATPase activities, suggesting this protein has an important role in viral RNA replication in virus-infected cells.