Recombinant Nonstructural Protein Research Articles

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.

Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Serologic Cross-reactivity of Murine Parvovirus Capsid Antigens.

Genomic sequence analysis of autonomous parvoviruses within the genus Protoparvovirus generates 2 groups that are principally of mouse origin: the minute virus of mice (MVM) strains (MVMp, MVMi, MVMc, MVMm) and the mouse parvovirus (MPV)-like strains (MPV-1, MPV-2, MPV-3, MPV-4, MPV-5, HaPV, LuIII). Baculovirus-expressed recombinant capsid protein (rVP2) from each of these 11 parvovirus strains were produced, purified, and demonstrated to form virus-like particles. Each rVP2 preparation was then used as antigen in a multiplex fluorescent immunoassay and to immunize 5 different strains of mice. Sera from immunized mice, mice experimentally monoinfected with various MVM or MPV isolates, and mice naturally infected with murine parvoviruses were evaluated with the multiplex fluorescent immunoassay rVP2 panel. Results for sera from immunized mice indicate that homologous antigen-antisera interactions produced the strongest seroreactivity. All MVM antigens were highly cross-reactive with heterologous MVM antisera, while more variability was observed in heterologous antigen-antisera reactions among the MPV-like strains. MPV-1, MPV-3, HaPV, and LuIII were highly cross-reactive with each other, MPV-2 and MPV-5 were highly cross-reactive with each other, and MPV-4 displayed modest cross-reactivity with certain MPV-like strains. Serologic cross-reactivity patterns similar to those in immunized mice were observed in mice experimentally infected with MVMp, MVMm, MPV-1, MPV-5, or HaPV, and in sera from mice naturally infected with MVM and MPV. Serologic cross-reactivity spectrums suggest a small panel of rVP2 antigens (MVM, MPV-1, MPV-2, MPV-4) combined with the generic murine parvovirus recombinant nonstructural protein 1 (rNS1) antigen are sufficient for qualitative detection of currently known MVM and MPV-like strains.

Expression of global consensus non-structural protein 1 (NS1) of dengue virus serotypes 1 and 2 in Nicotiana benthamiana

Dengue viruses, which consist of four distinct serotypes, belong to the Flavivirus family, one of the most important arthropod-borne pathogen groups for humans. Vietnam is among the tropical countries that have endemic dengue outbreaks, and in 2022, the number of infections as well as fatal cases reached a record high, causing a great economic burden to the public health system. Although all four dengue serotypes have been circulating in Vietnam, serotypes 1 and 2 have been dominating in the last few decades. Since most dengue clinical complications result from secondary infection with a different serotype from the first infection, prior knowledge of the proportion of a population having seropositive to a certain serotype is very crucial for public health policy makers. The absence of comprehensive and regular serological epidemiological surveys in Vietnam has impeded progress in surveillance and the implementation of effective preventive measures. To facilitate research into dengue treatment and diagnosis, we created two synthetic, codon-optimized genes encoding for global consensus non-structural protein 1 (NS1) from dengue serotypes 1 and 2. We expressed these genes in Nicotiana benthamiana using the agroinfiltration technique. Western blot using Dengue patients’ sera indicated that the target proteins were expressed, likely in both mono and multimeric forms. Our results indicated that N. benthamiana is a good alternative to other methods of producing recombinant NS1.

The adjuvant effect of polymuramil, a NOD1 and NOD2 agonist, differs when immunizing mice of different inbred lines with nonstructural hepatitis C virus (Flaviviridae: Hepacivirus)proteins and is synergistically enhanced in combination with pyrogenalum, a TLR4 agonist.

Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines. The aim of the work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum. BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response - by antigen-specific lymphocyte proliferation and by production of IFN-γ in vitro. Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN-γ in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins. Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.

Expression of recombinant dengue virus type 1 non-structural protein 1 in mammalian cells and preliminary assessment of its suitability to detect human IgG antibodies elicited by viral infection

In recent years dengue has become a rapidly growing public health problem worldwide, however, the availability of accurate and affordable diagnostic immunoassays is limited, partly due to the difficulty of producing large quantities of purified antigen. Non-structural protein 1 (NS1) has shown to be a good candidate for inclusion in diagnostic assays and for serosurveys, particularly in endemic countries as a prerequisite for vaccination. In this work the NS1 antigen derived from dengue virus type-1 (DENV1) was expressed in HEK293-T cells and purified by affinity chromatography. The recombinant protein was recovered properly folded as dimers, highly purified and with good yield (1.5 mg/L). It was applied as a serological probe in an indirect ELISA developed in this work to detect human IgG antibodies. Preliminary comparative performance values of 81.1% sensitivity and 83.0% specificity of the developed and preliminary validated iELISA, relative to a commercial kit were obtained, suggesting that the purified recombinant DENV1 NS1 antigen is suitable to detect IgG antibodies, indicative of past DENV infection.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection.

Extracellular vesicles (EVs), produced during viral infections, are of emerging interest in understanding infectious processes and host-pathogen interactions. EVs and exosomes in particular have the natural ability to transport nucleic acids, proteins, and other components of cellular or viral origin. Thus, they participate in intercellular communication, immune responses, and infectious and pathophysiological processes. Some viruses are known to hijack the cell production and content of EVs for their benefit. Here, we investigate whether two pathogenic flaviviruses i.e., Zika Virus (ZIKV) and Dengue virus (DENV2) could have an impact on the features of EVs. The analysis of EVs produced by infected cells allowed us to identify that the non-structural protein 1 (NS1), described as a viral toxin, is associated with exosomes. This observation could be confirmed under conditions of overexpression of recombinant NS1 from each flavivirus. Using different isolation methods (i.e., exosome isolation kit, size exclusion chromatography, Polyethylene Glycol enrichment, and ELISA capture), we showed that NS1 was present as a dimer at the surface of excreted exosomes, and that this association could occur in the extracellular compartment. This finding could be of major importance in a physiological context. Indeed, this capacity of NS1 to address EVs and its implication in the pathophysiology during Dengue or Zika diseases should be explored. Furthermore, exosomes that have demonstrated a natural capacity to vectorize NS1 could serve as useful tools for vaccine development.

Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the β-ladder domain.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the β-ladder domain (amino acid residues 178–273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.

Reverse Genetics System for Heartland Bandavirus: NSs Protein Contributes to Heartland Bandavirus Virulence.

Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

Detection of NS1 protein from dengue virus in excreta and homogenates of wild-caught Aedes aegypti mosquitoes using monoclonal antibodies.

Dengue fever is one of the most devastating infectious diseases worldwide. Development of methods for dengue virus (DENV) detection in mosquitoes to assess prevalence as a preliminary screen for entomological surveillance in endemic regions of DENV will certainly contribute to the control of the disease. A monoclonal antibody against the NS1 (nonstructural protein 1) viral protein was generated using recombinant NS1 protein and used to detect and analyze DENV in both excreta and total homogenates from Aedes aegypti mosquitoes. Results demonstrated expression of NS1 in excreta of DENV laboratory-infected mosquitoes and homogenates from field mosquitoes infected with DENV. The immunodetection method reported here represents a first-line strategy for assessing the prevalence of DENV in mosquitoes, for entomological surveillance in endemic regions of dengue. Detection of DENV prevalence in field mosquitoes could have an impact on vector surveillance measures to interrupt dengue transmission.

Nanobead-Based Screening Method for Antibody Pairing of Dengue Virus Nonstructural Protein-1.

Dengue fever is a classic mosquito viral disease. Dengue virus non-structural protein-1 as a membrane-associated homologous dimer anchored to the surface of infected cells and also secreted into the blood. The nonstructural protein-1 levels are related to disease severity, and the presence of nonstructural protein-1 secreted from cells to the serum of people infected with the dengue virus is an early marker of infection. Paired antibodies are key in the establishment of rapid detection technology. In this study, the prepared recombinant nonstructural protein-1 protein of dengue virus serotype 3 was purified by the prokaryotic expression, and prepared monoclonal antibodies by cell fusion. A method for paired antibody screening was established based on the N-hydroxy succinimide-nanobeads and the prepared monoclonal antibodies. A simple and rapid point-of-care system integrating the paired antibodies and lateral flow assay was established to verify the screened antibody pairs. The results confirmed that the antibody pair screening method based on N-hydroxy succinimide-nanobeads is feasible.

N130, N175 and N207 are N-linked glycosylation sites of duck Tembusu virus NS1 that are important for viral multiplication, viremia and virulence in ducklings

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.

Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1.

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

Specificity of NS1-based immunochromatographic tests for dengue virus with regard to the Zika virus protein

ObjectivesThis study was performed to determine whether Dengue virus (DENV) immunochromatographic tests can detect and differentiate nonstructural protein 1 (NS1) from each of the four DENV serotypes and do not cross-react with the Zika virus (ZIKV) NS1 protein. MethodsWe compared the specificity of six NS1-based DENV immunochromatographic tests (point of care) in the detection of NS1 proteins from each of the four DENV serotypes and ZIKV. The tests were performed with NS1 proteins produced in mammalian cells. Cross-reactivity was confirmed with a purified recombinant ZIKV NS1 protein and DENV+ or ZIKV+ human serum samples. ResultsCross-reaction was observed in 2 out of the 6 evaluated tests using cell culture supernatants containing NS1 protein of each tested virus. Cross-reactivity with ZIKV was confirmed with purified recombinant ZIKV NS1 produced in Escherichia coli. Further analyses with serum samples collected from DENV+ or ZIKV+ patients confirmed the cross-reactivity with ZIKV protein in 2 tests. ConclusionsThe detection of the NS1 protein is the basis for several commercially available serological DENV diagnostic tests. The present results emphasize the relevance of testing specificity of presently available NS1-based DENV serological tests and the need of adjustments of tests that cross-react with the ZIKV protein. Our results are particularly relevant for regions where both viruses are endemically found, as in the case of Brazil.

Optimization of recombinant Zika virus NS1 protein secretion from HEK293 cells

Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50 nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of ∼10 mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.

A nonstructural 2B protein of enterovirus A71 increases cytosolic Ca2+ and induces apoptosis in human neuroblastoma SH-SY5Y cells

Enterovirus A71 (EV-A71) is one of the causative agents causing the hand-foot-mouth disease which associated with fatal neurological complications. Several sporadic outbreaks of EV-A71 infections have been recently reported from Asia-Pacific regions and potentially established endemicity in the area. Currently, there is no effective vaccine or antiviral drug for EV-A71 available. This may be attributable to the limited information about its pathogenesis. In this study, the recombinant nonstructural 2B protein of EV-A71 was successfully produced in human neuroblastoma SH-SY5Y cells and evaluated for its effects on induction of the cell apoptosis and the pathway involved. The EV-A71 2B-transfected SH-SY5Y cells showed significantly higher difference in the cell growth inhibition than the mock and the irrelevant protein controls. The transfected SH-SY5Y cells underwent apoptosis and showed the significant upregulation of caspase-9 (CASP9) and caspase-12 (CASP12) genes at 3- and 24-h post-transfection, respectively. Interestingly, the level of cytosolic Ca2+ was significantly elevated in the transfected SH-SY5Y cells at 6- and 12-h post-transfection. The caspase-9 is activated by mitochondrial signaling pathway while the caspase-12 is activated by ER signaling pathway. The results suggested that EV-A71 2B protein triggered transient increase of the cytosolic Ca2+ level and associated with ER-mitochondrial interactions that drive the caspase-dependent apoptosis pathways. The detailed mechanisms warrant further studies for understanding the implication of EV-A71 infection in neuropathogenesis. The gained knowledge is essential for the development of the effective therapeutics and antiviral drugs.

Remodeling of the Actin Network Associated with the Non-Structural Protein 1 (NS1) of West Nile Virus and Formation of NS1-Containing Tunneling Nanotubes.

The cellular response to the recombinant NS1 protein of West Nile virus (NS1WNV) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1WNV: (i) the exogenous secreted form, sNS1WNV, added to the extracellular milieu; and (ii) the endogenous NS1WNV, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1WNV varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1WNV to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1WNV and actin network remodeling were dependent on cell type. In the three cell types, NS1WNV-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1WNV proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1WNV and the viral envelope glycoprotein EWNV were also observed in WNV-infected Vero E6 cells.