Resuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virus

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37 degrees C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28 degrees C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.     

Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.     

NS3 protease of Langat tick-borne flavivirus cleaves serine protease substrates

A DNA fragment of 163 bp containing 11 GGA repeats formed two-end positioned mononucleosomes as efficiently as that of CTG repeats. However, the rotational positioning of the GGA fragment was weak because clear DNase I cleavage patterns with 10-base periodicity were not seen near the center of the GGA fragment but were detected in the entire region of the CTG fragment. Incubation of the GGA mononucleosomes with the same fragment provided the DNA-DNA complex, which had been shown by using naked DNA fragments. DNase I digestion of the complex exhibited protection in the GGA repeats and in flanking sequences of about 30 bp at both sides, suggesting that both the repeat and flanking regions were involved in the association. Interestingly, histone H1, which enhanced DNA-DNA association on naked DNA, did not affect the complex formation on mononucleosomes. These results imply that GGA microsatellites in genomes could associate with one another at multiple sites and that the association may play a role in functional organization of higher order chromatin architecture.     

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.     

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

A model of the tick-borne encephalitis virus envelope protein E is presented that contains information on the structural organization of this flavivirus protein and correlates epitopes and antigenic domains to defined sequence elements. It thus reveals details of the structural and functional characteristics of the corresponding protein domains. The localization of three antigenic domains (composed of 16 distinct epitopes) within the primary structure was performed by (i) amino-terminal sequencing of three immunoreactive fragments of protein E and (ii) sequencing the protein E-coding regions of seven antigenic variants of tick-borne encephalitis virus that had been selected in the presence of neutralizing monoclonal antibodies directed against the E protein. Further information about variable and conserved regions was obtained by a comparative computer analysis of flavivirus E protein amino acid sequences. The search for potential T-cell determinants revealed at least one sequence compatible with an amphipathic alpha-helix which is conserved in all flaviviruses sequenced so far. By combining these data with those on the location of disulfide bridges (T. Nowak and G. Wengler, Virology 156:127-137, 1987) and the structural characteristics of epitopes, such as dependency on conformation or on intact disulfide bridges or both, a model was established that goes beyond the location of epitopes in the primary sequence and reveals features of the folding of the polypeptide chain, including the generation of discontinuous protein domains.     

A tentative model of formation of structural proteins of tick-borne encephalitis virus (flavivirus)

The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [³⁵S]-methionine or formyl[³⁵S]methionyl-tRNA_f^Met as substrates, and the [³⁵S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed.     

Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus

A new vaccination principle against flaviviruses, based on a tick-borne encephalitis virus (TBEV) self-replicating noninfectious RNA vaccine that produces subviral particles, has recently been introduced (R. M. Kofler, J. H. Aberle, S. W. Aberle, S. L. Allison, F. X. Heinz, and C. W. Mandl, Proc. Natl. Acad. Sci. USA 7:1951-1956, 2004). In this study, we evaluated the potential of the self-replicating RNA vaccine in mice in comparison to those of live, attenuated vaccines and a formalin-inactivated whole-virus vaccine (ImmunInject). For this purpose, mice were immunized using gene gun-mediated application of the RNA vaccine and tested for CD8+ T-cell responses, long-term duration, neutralizing capacity, and isotype profile of specific antibodies and protection against lethal virus challenge. We demonstrate that the self-replicating RNA vaccine induced a broad-based, humoral and cellular (Th1 and CD8+ T-cell response) immune response comparable to that induced by live vaccines and that it protected mice from challenge. Even a single immunization with 1 microg of the replicon induced a long-lasting antibody response, characterized by high neutralizing antibody titers, which were sustained for at least 1 year. Nevertheless, it was possible to boost this response further by a second injection with the RNA vaccine, even in the presence of a concomitant CD8+ T-cell response. In this way it was possible to induce a balanced humoral and cellular immune response, similar to infection-induced immunity but without the safety hazards of infectious agents. The results also demonstrate the value of TBEV replicon RNA for inducing protective long-lasting antiviral responses.     

Synthesis and membrane-dependent processing of a precursor of tick-borne encephalitis virus (flavivirus) structural proteins in cell-free systems

Conditions that permitted cell-free synthesis of at least one of the non-structural, in addition to two-structural, polypeptides of tick-borne encephalitis virus have been found. The time course of accumulation of virus-specific polypeptides in extracts of Krebs-2 cells and reticulocyte lysates as well as the peptide maps of the products synthesised were studied. A model of generation of viral structural polypeptides has been proposed, according to which a common precursor of these proteins while in a nascent form, is processed in a membrane-dependent reaction into a C-terminal segment, corresponding to the polypeptide moiety of envelope glycoprotein E, and an N-terminal segment, doublet p36/33. Subsequently, an N-terminal segment, corresponding to the core polypeptide C, is cleaved off from p36/33. The remaining C-terminal segment of p36/33 is possibly a precursor of the membrane polypeptide M. The translational strategy of flaviviruses is compared to that of other positive-stranded RNA viruses.     

The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization.

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.     

Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions.

Recombinant subviral particles (RSPs) obtained by coexpression of the envelope (E) and premembrane (prM) proteins of tick-borne encephalitis virus in COS cells (S. L. Allison, K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz, J. Virol. 69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [14C]choline but not [3H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers. The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.     

蜱传脑炎病毒对人单核细胞的致病性北大核心CSCD

【目的】确定蜱传脑炎病毒(Tick-born encephalitis virus,TBEV)对人单核细胞的感染性及对其复制增殖的影响。【方法】用蜱传脑炎病毒感染单核细胞THP-1,观察细胞病变情况。取不同时间点的细胞培养上清,测定病毒滴度,并用Real Time RT-PCR方法检测病毒核酸;用流式细胞法检测细胞感染率,以确定TBEV在THP-1细胞中的复制增殖情况;同时进行细胞活力检测,以确定TBEV感染后THP-1细胞的变化。【结果】TBEV病毒感染THP-1细胞后,可进行复制增殖,流式细胞法可检测到细胞内的病毒,感染病毒后的单核细胞活力显著降低。【结论】TBEV可在单核细胞THP-1中复制增殖,并可造成细胞活力的显著降低,提示单核细胞可能在TBEV感染机体并扩散至各组织器官过程中发挥了重要作用。     

Unexpected altered specificity is responsible for St. Louis encephalitis virus recombinant protease autoproteolysis

We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.     

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。     

Comparison of the specificity of polyclonal antibodies to the tick-borne encephalitis virus in immunoenzyme analysis and immunoblotting

The present study deals with the optimization of the conditions of immunoblotting (IB) and the enzyme-linked immunosorbent assay (ELISA) with the use of mouse polyclonal immunoglobulins to tick-borne encephalitis virus. In contrast to ELISA, the results of IB depended, to a great extent, on the composition and pH of blocking buffer mixtures. In some cases IB permitted the detection of a heretofore unknown virus-specific polypeptide with a molecular weight of 58-60 KD. The results of the study lead to the conclusion on the impossibility of the direct transfer of data concerning the specificity of individual preparations of immunoglobulins in ELISA or IB due to differences in information.     

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.     

Characterization of Rabensburg virus, a flavivirus closely related to West Nile virus of the Japanese encephalitis antigenic group

Rabensburg virus (RABV), a Flavivirus with ～76% nucleotide and 90% amino acid identity with representative members of lineage one and two West Nile virus (WNV), previously was isolated from Culex pipiens and Aedes rossicus mosquitoes in the Czech Republic, and phylogenetic and serologic analyses demonstrated that it was likely a new lineage of WNV. However, no direct link between RABV and human disease has been definitively established and the extent to which RABV utilizes the typical WNV transmission cycle is unknown. Herein, we evaluated vector competence and capacity for vertical transmission (VT) in Cx. pipiens; in vitro growth on avian, mammalian, and mosquito cells; and infectivity and viremia production in birds. RABV infection and replication only were detected on mosquito cells. Experimentally inoculated birds did not become infected. Cx. pipiens had poor peroral vector competence and a higher VT rate as compared to US-WNV in Cx. pipiens. As a result, we postulate that RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses.     

Isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particles

Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses.