Expression of gene coding for the NS1 protein of the tick-borne encephalitis virus in Escherichia coli cell

Two possible forms of tick-borne encephalitis virus (TBE) gene NS1 (called NS1' and NS1) were constructed using two overlapping cDNA-fragments of TBE genome and synthetic DNA fragments. This genes were expressed in E. coli cells in expression vector pUR290 as individual proteins or fusion with bacterial beta-galactosidase. The proteins NS1 (Mw. 39 kDa), beta-galactosidase-NS1' (Mw. 162 kDa) and beta-galactosidase-NS1 (Mw. 155 kDa) were effectively synthesized under the Plgc-promoter induction conditions. Expression of NS1' gene results in the formation of two virus-specific proteins (Mw. 46 and 44 kDa). All bacterial analogs of NS1 protein fixed monoclonal and polyclonal antibodies specific to viral NS1.     

Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells]

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.     

A short form of the tick-borne encephalitis virus NS3 protein.

Using monoclonal antibodies to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were revealed in TBE-infected mammalian cells: a full-length form (69 kDa) and a short form (49 kDa) which has not been observed before and was called NS3'. Recombinant plasmids were constructed and various fragments of the TBE NS3 gene were expressed in rabbit reticulocyte lysate. By analyzing immune precipitates of 35S-labeled translation products, we could monitor and localize internal cleavage of NS3, due to which the NS3' protein was generated.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16-to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20?ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.     

Prevalence of tick-borne encephalitis virus antibodies in dogs from Denmark

Background  

Large regions of central and eastern Europe are recognized as areas where tick-borne encephalitis virus (TBEV) is endemic, including countries neighbouring Denmark. It is therefore timely and relevant to determine if TBEV infections occur in Denmark. This study investigates the presence of antibodies against TBEV in a cross-section of the Danish canine population to assess the level of exposure to TBEV and possibly identify TBEV microfoci in Denmark.     

森林脑炎病毒分子生物学研究进展

森林脑炎(TBE)病毒属黄病毒科,基因姐RNA含有单个开放阅读框架,5′端编码病毒的结构蛋白,3′端编码非结构蛋白。翻译成聚蛋白后,通过细胞和病毒编码的蛋白酶裂解产生单个的病毒蛋白。成熟的病毒是由两个相关的E和M膜蛋白脂质包膜所包围的立体对称的核衣壳组成。包膜E蛋白在病毒的感染周期中对细胞的识别和穿入细胞具有极其重要的功能,同时E蛋白诱导保护性的免疫反应,E蛋白内某一位点单个氨基酸的改变可引起病毒毒力的改变。因此,对TBE病毒分子生物学的研究有助于了解病毒与宿主细胞相互作用的机理,为病毒感染的特异性诊断、疫苗的研制和抗病毒药物的设计提供理论依据。     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits

The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.     

Characteristics of tick-borne encephalitis virus cirulating in Poland.

An attempt of chacteristization of tick-borne encephalitis virus strains, isolated in Poland in 1955--1970, was based on the determination and analysis of neurovirulence for white mice at intracerbral and subcutaneous virus inoculation, neuroinvasion, antigenic properties, cytopathogenic abilities, susceptibility to nonspecific hemagglutination inhibitors, susceptibility to temperature of 50degree and to 2M urea. Strains isolated in different regions of the country showed certain differentiation in pathogenic and antigenic properties as well in susceptibility to nonspecific inhibitors.     

Parameters affecting gene expression from the Pm promoter in gram-negative bacteria

The Pm promoter inserted chromosomally or in broad-host-range replicons based on plasmid RSF1010 or RK2 are useful systems for both high- and low-level expression of cloned genes in several gram-negative bacterial species. The positive Pm regulator XylS is activated by certain substituted benzoic acid derivatives, and here we show that these effectors induce expression of Pm at similar relative ranking levels in both Escherichia coli and Pseudomonas aeruginosa However, the kinetics of expression was not the same in the two organisms. Different carbon sources and dissolved oxygen levels displayed limited effects on expression, but surprisingly the pH of the growth medium was found to be of major importance. By combining the effects of genetic and environmental parameters, expression from Pm could be varied over a ten-thousand- to a hundred-thousand-fold continuous range, and as an example of its applications we showed that Pm can be used to control the xanthan biosynthesis in Xanthomonas campestris.     

Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus

It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.     

Structural modifications of macrophages initiated by tick-borne encephalitis virus

Macrophages are cells of natural immunity and play a key role in pathogenesis of viral infections. Results of ultrastructural research on macrophages infected with tick-borne encephalitis virus (TBEV), an agent that causes dangerous infections affecting nervous system in human beings and belongs to the Flaviviridae family, were shown here. Using virology methods, it was ascertained that the TBEV is consumed by macrophages and multiplies in them. Ultrastructural research showed that the virus penetrates into the cytoplasm by means of local plasmalemma lysis and newly synthesized virus particles escape from the cell by the same path. At the same time, induration of the perinuclear space of cytoplasm was observed, where ribosomes, microfilaments, ribonucleoprotein threats, and virus-specific structures, namely, nucleocapsids, tube formations, and viroplasts, were found in large quantities. On the surface of viroplasts, newly synthesized virus particles were visualized. Thus, evidence was presented that microphages can play certain role in spreading of TBEV and are the target of the virus. Like active antigen-presenting cells, such macrophages can modulate the protective response of an organism and affect the pathogenesis of tick-borne encephalitis.     

In vitro activity of luromarin against tick-borne encephalitis virus

Luromarin, extracted from the sea alga Zostera asiatica, showed no cytotoxic activity when incubated with the SPEV cell culture and was characterized by virulicidal properties against highly pathogenic strain of TBE virus and the ability to inhibit the virus replication at the early stages of the infection.     

Proteolytic activation of tick-borne encephalitis virus by furin.

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.