N-糖基化对乙型脑炎病毒prME和NS1基因免疫效果影响的研究

prME和NS1为乙型脑炎病毒两个主要的免疫保护蛋白,且均为N-糖蛋白。为研究N-糖基化对乙型脑炎病毒免疫保护的作用,本研究用PCR介导的定点突变方法,分别消除乙型脑炎病毒prME和NS1基因的不同N-糖基化位点,并构建了prME和NS1突变基因的真核表达质粒。将质粒免疫四周龄雌性小白鼠,经两次免疫后,采集血清检测体液免疫反应,最后对小鼠用强毒进行攻击,观察并记录免疫保护力。研究结果显示,与野生型prME基因免疫组相比,消除单个糖基化位点后prME基因诱导的ELISA抗体、中和抗体和免疫保护力均略有升高,而同时消除两个糖基化位点的则会降低。NS1基因消除单个糖基化位点后保护率高达到100%,但消除两个糖基化位点后则免疫保护率略有降低(75%)。通过本研究证明,N-糖基化在维系乙型脑炎病毒prME和NS1蛋白的免疫保护中具有重要的作用,单个糖基化的缺失可增强蛋白的免疫原性,而两个糖基都缺失后,则造成了免疫效率的降低。     

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ‐induced responses, for example, expression of IFN‐stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress‐induced‐phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca²⁺ levels while inhibiting nuclear translocalization of STAT1 and NFAT‐1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca²⁺/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5‐mediated inhibition of IFNβ‐induced responses, for example, IFN‐sensitive response element driven luciferase, STAT1‐dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5‐expressing cells in response to IFNβ. Results prove Ca²⁺, calreticulin, and calcineurin involvement in STAT1‐mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.     

Japanese Encephalitis Virus NS1′ Protein Antagonizes Interferon Beta Production

Japanese encephalitis virus(JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a52-amino acid C-terminal extension of NS1, is generated with a-1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected(rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus(pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type Ⅰ interferon(IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-b and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.     

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.     

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.     

Inoculation of plasmids encoding Japanese encephalitis virus PrM-E proteins with colloidal gold elicits a protective immune response in BALB/c mice

We established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti-JEV antibodies and a protective response against JEV challenge in BALB/c mice. When we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit stronger and more sustained neutralizing immune responses than intramuscular or intraperitoneal injection. After being inoculated twice, mice were found to resist challenge with 100,000 times the 50% lethal dose (LD(50)) of JEV (Beijing-1 strain) even when immunized with a relatively small dose of 0.5 micro g of plasmid DNA. Protective passive immunity was also observed in SCID mice following transfer of splenocytes or serum from plasmid DNA- and colloidal gold-immunized BALB/c mice. The SCID mice resisted challenge with 100 times the LD(50) of JEV. Analysis of histological sections detected expression of proteins encoded by plasmid DNA in the tissues of intravenously, intradermally, and intramuscularly inoculated mice 3 days after inoculation. DNA immunization with colloidal gold elicited encoded protein expression in splenocytes and might enhance immune responses in intravenously inoculated mice. This approach could be exploited to develop a novel DNA vaccine.     

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

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

乙型脑炎病毒NS2A-C60A位点突变对其生物学特性影响研究*

目的:旨在探索Ⅰ型日本乙型脑炎病毒传代致弱后基因组突变NS2A-C60A对乙脑病毒生物学特性的影响。方法:首先通过对传代致弱及原始乙脑毒株基因组序列进行测序比对、结构预测分析并利用Western blotting(WB)确定了目标研究位点NS2A-C60A;然后使用反向遗传定点突变技术构建拯救了包含NS2A-C60A单点突变的病毒株;最后利用噬斑形态观察、生长曲线、双萤光素酶分析,WB以及炎性因子检测和动物实验研究了该单点突变对于乙脑病毒生物学特性的影响。结果:首次研究发现Ⅰ型乙脑病毒传代致弱会导致NS1'蛋白表达的显著下降以及可能的相关位点NS2A-C60A,并成功拯救获得了NS2A-C60A单点突变毒株rJEV-C60A,研究发现NS2A-C60A突变对乙脑病毒的生长特性及噬斑形成没有显著影响,但是能够显著降低乙脑病毒NS1'蛋白的表达,并且该位点突变能够轻微阻碍乙脑病毒对细胞炎性因子表达的抑制,动物实验结果显示NS2A-C60A点突变病毒与原毒株具有相似的神经毒力,说明该位点突变不是影响乙脑病毒毒力致弱的关键位点。结论:新发现的NS2A-C60A位点突变能够显著减少乙脑病毒NS1'蛋白的表达,但是对其增殖、诱导炎症及神经毒力等生物学特性没有显著影响。     

Productive Homologous and Non-homologous Recombination of Hepatitis C Virus in Cell Culture

Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.