Japanese Encephalitis Virus Proteins Research Articles

Antibodies Against Refolded Recombinant Envelope Protein (Domain III) of Japanese Encephalitis Virus Inhibit the JEV Infection to Porcine Stable Kidney Cells

Japanese encephalitis virus (JEV) is a mosquito-borne viral zoonosis of public health importance. Global efforts have been made towards development of vaccine for prevention of Japanese encephalitis. The envelope protein of JEV is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing antibody response in hosts. Here we report that the antibodies raised against refolded domain III of envelope protein of JEV neutralize the JE virus and inhibit the JEV infection to Porcine Stable Kidney (PS) cells. A reverse transcriptase-PCR amplified gene encoding domain III of JEV envelope protein was cloned into pET28a+ vector and over expressed in E. coli. The recombinant JEV-DIII protein was purified by affinity chromatography under denaturing conditions. The rJEV-DIII was refolded by oxido-redux shuffle and purified to homogeneity by ion-exchange chromatography. Refolded rJEV-DIII was characterized using biochemical and biophysical methods. The polyclonal antibodies were raised against in vitro refolded rJEV-DIII protein in BALB/c mice with Freunds adjuvant. Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80 as against 60.5% neutralization capacity with the same dilution of serum raised against denatured rJEV-DIII. The method of expression and purification of biologically functional rJEV-DIII protein described in this study may help in better understanding the biology of JE virus and the development of better vaccine candidate. Since the expression system uses E. coli as the heterologous host, the process is easy and amenable to inexpensive scale-up.

Structure-based virtual screening for novel inhibitors of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase

Japanese encephalitis (JE) is a significant cause of human morbidity and mortality throughout Asia and Africa. Vaccines have reduced the incidence of JE in some countries, but no specific antiviral therapy is currently available. The NS3 protein of Japanese encephalitis virus (JEV) is a multifunctional protein combining protease, helicase and nucleoside 5′‐triphosphatase (NTPase) activities. The crystal structure of the catalytic domain of this protein has recently been solved using a roentgenographic method. This enabled structure‐based virtual screening for novel inhibitors of JEV NS3 helicase/NTPase. The aim of the present research was to identify novel potent medicinal substances for the treatment of JE. In the first step of studies, the natural ligand ATP and two known JEV NS3 helicase/NTPase inhibitors were docked to their molecular target. The refined structure of the enzyme was used to construct a pharmacophore model for JEV NS3 helicase/NTPase inhibitors. The freely available ZINC database of lead‐like compounds was then screened for novel inhibitors. About 1 161 000 compounds have been screened and 15 derivatives of the highest scores have been selected. These compounds were docked to the JEV NS3 helicase/NTPase to examine their binding mode and verify screening results by consensus scoring procedure.

The prM-independent packaging of pseudotyped Japanese encephalitis virus

As noted in other flaviviruses, the envelope (E) protein of Japanese encephalitis virus (JEV) interacts with a cellular receptor and mediates membrane fusion to allow viral entry into target cells, thus eliciting neutralizing antibody response. The formation of the flavivirus prM/E complex is followed by the cleavage of precursor membrane (prM) and membrane (M) protein by a cellular signalase. To test the effect of prM in JEV biology, we constucted JEV-MuLV pseudotyped viruses that express the prM/E protein or E only. The infectivity and titers of JEV pseudotyped viruses were examined in several cell lines. We also analyzed the neutralizing capacities with anti-JEV sera from JEV-immunized mice. Even though prM is crucial for multiple stages of JEV biology, the JEV-pseudotyped viruses produced with prM/E or with E only showed similar infectivity and titers in several cell lines and similar neutralizing sensitivity. These results showed that JEV-MuLV pseudotyped viruses did not require prM for production of infectious pseudotyped viruses.

Expression of Japanese encephalitis virus envelope protein in transgenic tobacco plants

The virus envelope (E) protein of Japanese encephalitis virus induces virus-neutralizing antibodies and is therefore a potential vaccine antigen. In a mammalian system, co-expression of another viral structural protein prM is necessary for proper expression and folding of E protein. Transgenic tobacco plants were produced carrying JEV cDNA encoding prM and E proteins under the control of the CaMV 35S promoter. E protein, however, was not detectable in these plants. In vitro translation studies showed that the presence of the prM sequence inhibited transgene expression in the plant system. Accordingly, JEV E protein could be expressed in transgenic tobacco plants only without the prM protein.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen‐specific but non‐neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a model antigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection. Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

3 molecule as putative Japanese encephalitisvirus receptor

：Objective To isolate andidentify the putative Japanese encephalitis virus (JEV) re-ceptors from C6/36 and Verocells. Methods Molecules binding with JEV were isolated from C6/36 and Vero cells byco-immunoprecipitation (Co-IP) approach, identified by mass spectrometry, and detected byWestern blot. The location of putative JEV receptor on cells membrane and the binding withJEV were ob-served by laser scanning confocal microscopy (LCM). Results Several moleculesbinding with JEV were isolated from C6/36 and Vero cells by Co-IP, and only one moleculewas identified as heat shock cognate 70 (HSC70) by mass spectrometry. Antibody againstHSC'70 was able to detect a 74 x 103 protein isolated by Co-IP from C6/36 and Vero cellsmembrane in Western blot assays. It was observed by LSCM that when JEV attached on thesurface of C6/36 cells, JEV and HSCT0 protein were co-localization. Conclusion 74 x 103molecular identified as HSC70 protein from C6/36 cells may be JEV receptor.

Isolation and preliminary identification of 74×103 molecule as putative Japanese encephalitis virus receptor

Objective To isolate and identify the putative Japanese encephalitis virus (JEV) re-ceptors from C6/36 and Vero cells. Methods Molecules binding with JEV were isolated from C6/36 and Vero cells by co-immunoprecipitation (Co-IP) approach, identified by mass spectrometry, and detected by Western blot. The location of putative JEV receptor on cells membrane and the binding with JEV were ob-served by laser scanning confocal microscopy (LCM). Results Several molecules binding with JEV were isolated from C6/36 and Vero cells by Co-IP, and only one molecule was identified as heat shock cognate 70 (HSC70) by mass spectrometry. Antibody against HSC'70 was able to detect a 74 ×103 protein isolated by Co-IP from C6/36 and Vero cells membrane in Western blot assays. It was observed by LSCM that when JEV attached on the surface of C6/36 cells, JEV and HSCT0 protein were co-localization. Conclusion 74 x 103 molecular identified as HSC70 protein from C6/36 cells may be JEV receptor. Key words: Japanese encephalitis virus ; Virus receptor ; Co-immunoprecipitation ; C6/36 cells ; Vero cells ;

Erratum: Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice Yaoming Li Jing Ye Shengbo Cao Shaobo Xiao Qian Zhao Xueqin Liu Meilin Jin Huanchun Chen

Erratum: Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice Yaoming Li Jing Ye Shengbo Cao Shaobo Xiao Qian Zhao Xueqin Liu Meilin Jin Huanchun Chen

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.

Cloning and expression of domain III of the envelope gene of Japanese encephalitis virus: Evaluation for early clinical diagnosis by IgM ELISA

Japanese encephalitis virus (JEV) is the single largest cause of childhood viral encephalitis in the world with an estimated 50,000 cases and 10,000 deaths annually. The laboratory diagnosis is based essentially on IgM ELISA owing to low transient viremia making virus isolation difficult. In addition the requirement of cerebrospinal fluid (CSF) sample for confirmatory molecular diagnosis by reverse transcription-polymerase chain reaction (RT-PCR) makes IgM ELISA the test of choice for early clinical diagnosis. The development and evaluation of a highly sensitive and specific IgM ELISA using the recombinant domain III envelope protein (rJEV-DIII) for rapid, early and accurate diagnosis of JEV is reported in the present study. The gene coding for the envelope protein of JEV was cloned and expressed in pET 30a vector followed by purification of recombinant protein by affinity chromatography. An indirect IgM microplate ELISA using purified rJEV-DIII was optimized that had no reactivity with healthy persons. The comparative evaluation accomplished with the JE-Dengue IgM Combo ELISA (PanBio, Brisbane, Australia) and JEV Chex (XCyton Diagnostic Ltd., Bangalore, India) ELISA kits, by subjecting 120 acute phase of clinical samples revealed more than 95% accordance. The rJEV-DIII ELISA and the PanBio ELISA were found to have a sensitivity and specificity of 98% and 96%, respectively. The compared positivity of the rJEV-DIII ELISA and SYBR green-I based real-time RT-PCR assay in CSF samples revealed higher positivity. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. The recombinant domain III envelope protein based JEV specific ELISA will be useful for rapid screening of large numbers of clinical samples in endemic areas during outbreaks.

Identification of a virus-specific and conserved B-cell epitope on NS1 protein of Japanese encephalitis virus

NS1 protein of Japanese encephalitis virus (JEV) is an important non-structural protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool, but the epitopes on NS1 of JEV have not been identified. For epitope mapping, in this study, a series of 51 partially overlapping fragments covering entire NS1 protein were expressed with a GST-tag and then screened by a monoclonal antibody (mAb). Through enzyme-linked immunosorbent assay (ELISA), linear epitope-containing fragment, the overlapping region of NS1-18 and NS1-19 (residues 145–152), was located. Then a set of peptides derived from that overlapping region with deletions were expressed and subjected to ELISA and Western blot for further mapping purpose. Results indicated that the motif of 146EHARW 150 is the minimal unit of the linear epitope recognized by that monoclonal antibody (mAb). Western blot showed that this epitope could be recognized by JEV-positive serum from pigs. Furthermore, it was found that the epitope is highly conserved among JEV strains through sequence alignments analysis. Notably, none of the homologous regions on NS1 proteins of other flavivirus could react with the mAb when they were tested for cross-reactivity, suggesting the potential clinical application of this epitope in differential diagnosis.

Withdrawn: Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice

Japanese encephalitis (JE) is a serious infection disease throughout southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a matter of high priority. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was described as an attractive gene-delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed recombinant pseudotype baculovirus encoding the Japanese encephalitis virus (JEV) envelope (E) protein and demonstrated that it could elicit high protective immunity in mice. Recombinant pseudotype baculovirus (BV-G-E) was generated by inserting JEV E gene fragment into pFastBac-VSV/G vector. BALB/c mice were immunized with BV-G-E and challenged with JEV wild-type strain. The neutralization antibody, interferon (IFN)-gamma expression and release, and survival rate were analysed and compared with the group of immunized with inactivated vaccine and DNA vaccine (pc-E) encoding the same gene of JEV. We demonstrated that intramuscular injections of BV-G-E at various doses into mice produced higher levels of JEV-specific neutralizing antibodies, IFN-gamma and better protective efficacy against a lethal challenge with JEV than that of pc-E. Furthermore, BV-G-E could elicit a higher level cellular immunity response and provide equal protective efficacy against JEV challenge compared to inactivated vaccine. Our data demonstrate that BV-G-E elicited higher levels of protective immunity than DNA vaccine and that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generations of vaccines against JEV infection.

Recombinant core proteins of Japanese encephalitis virus as activators of the innate immune response

Nitric oxide (NO) has been shown to suppress Japanese encephalitis virus (JEV) RNA synthesis, viral protein accumulation, and virus release from infected cells. In this article, the potential viral structural proteins as the activators of NO product were studied at the molecular level. First, the genomic region encoding the JEV structural proteins was cloned into a prokaryotic expression vector pET for high-level expression. After purification, these JEV recombinant proteins were added to macrophages to examine the productions of NO and pro-inflammatory mediators. In this study, the recombinant core protein, but not envelope (E), could trigger NO and pro-inflammatory mediators (TNF-alpha, IL-6, and IL-12) productions on macrophages. And their effects were about 85-95% relative to LPS-stimulated macrophages in a dose-dependent manner. Meanwhile, the rCore-2D could up regulate promoters of IL-8 and TNF-alpha via EGFP expression in reporter plasmid (IL-8p-EGFP and TNF-alphap-EGFP)-transfected cells by flow cytometric analysis. These results suggest that JEV core protein could regulate pro-inflammatory mediators and NO production, and may play a crucial role in the innate immunity for the host to restrict the initial stage of JEV infection.

Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice

Japanese encephalitis (JE) is a serious infection and disease in southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a high-priority target in the world. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) was described as an attractive gene delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed a recombinant pseudotype baculovirus encoding the JEV envelope (E) protein and demonstrated that it could elicit high protective immunity in mice. Recombinant pseudotype baculovirus (BV-G-E) was generated by inserting JEV E gene fragment into pFastBac-VSV/G vector. BALB/c mice were immunized with BV-G-E and challenged with JEV wild-type strain. The neutralization antibody, interferon (IFN)-gamma expression and release, and survival rate were analysed and compared with the group of immunized with inactivated vaccine and DNA vaccine (pc-E) encoding the same gene of JEV. We demonstrated that intramuscular injections of BV-G-E at various doses into mice produced higher levels of JEV-specific neutralizing antibodies, IFN-gamma and better protective efficacy against a lethal challenge with JEV than that of pc-E. Furthermore, BV-G-E could elicit a higher level of cellular immunity response and provide equal protective efficacy against JEV challenge compared to inactivated vaccine. Our data demonstrate that BV-G-E elicited higher levels of protective immunity compared to DNA vaccine and that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generations of vaccines against JEV infection.

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [ Macaca mulatta

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys ( Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1 mg plasmid DNA given IM, or 5 μg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5 mg DNA given IM or 5 μg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.

Functional assignment to JEV proteins using SVM.

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Japanese encephalitis protein vaccine candidates expressing neutralizing epitope and M.T hsp70 induce virus-specific memory B cells and long-lasting antibodies in swine

Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE protein vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids are based on a prokaryotic vector pET-32a(+). One plasmid, designated pET-32a(+)-epitope, encode a cassette consisting of a neutralizing epitope on envelope (E) protein of JE virus, whereas the other plasmid, designated pET-32a(+)-epitope-hsp70, express the fusion protein of the epitope and M.T hsp70. Some differences were detected in the immunogenicity of these two proteins in swine. Swine immunized twice with 2000 pmol of the neutralizing epitope or the fusion protein developed neutralizing antibody titers of respectively, 154 and 300, and anti-neutralizing epitope antibody titers of 10 4.25 and 10 6.0 by 3 weeks after the second immunization. In addition, swine immunized with the neutralizing epitope emulsified with adjuvant S206 or with imported mineral oil and Tween-80 induced neutralizing antibody titers of 196 and 244, and anti-neutralizing epitope antibody titers of 10 5.25 or 10 5.6 at the same time point. However, swine administered two doses of a commercial JE vaccine (attenuated virus preparation; JEV SA14-14-2 strain) developed less favorable antibody responses with neutralizing antibody titer 40 and anti-neutralizing epitope antibody titers 10 3.7. The anamnestic response was followed by monitoring titers 1 week after boosting with a viral antigen; swine immunized twice with the fusion protein showed a 177-fold increase in anti-neutralizing epitope titer, indicating a strong recall of the antibody response. The animals maintained detectable levels of anti-neutralizing epitope antibody for at least 105 days after two immunizations, indicating that these four protein antigens are able to stimulate virus-specific memory B cells and long-lasting antibodies at higher levels than is achieved using a current commercial attenuated JEV vaccine. The group immunized with the epitope fused to M.T hsp70 made the strongest proliferation of lymphocytes. Through the assay of the amount of interferon (IFN)-γ and interleukin (IL)-4 in the serum, swine immunized with the fusion protein increased IFN-γ in the serum which showed that M.T hsp70 potentiated Th1 immune response.

A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice

The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N(15)-X(16)-T(17), which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substituting for N(15) and/or T(17) and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an approximately 20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

A strong endoplasmic reticulum retention signal in the stem–anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles

Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem–anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170–180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem–anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem–anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.

Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 Å

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 Å resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly 199, Lys 200 and Thr 201), in addition to the polar residues within the NTP-binding pocket (Gln 457, Arg 461 and Arg 464), and also Arg 458 in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys 200 was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.