Purification of the structural and nonstructural proteins of St. Louis encephalitis virus.

We have developed a procedure for purifying both the structural and nonstructural proteins of flaviviruses from lysates of infected cell cultures. The procedure involves: immunoprecipitation to concentrate viral proteins and eliminate most of the cellular proteins, preparative polyacrylamide gel electrophoresis to separate the viral proteins, and hydroxyapatite chromatography, which eliminates most of the unlabeled cellular protein. This procedure offers an improvement over previous purification schemes in that there is no loss of viral proteins after the immunoprecipitation step, any combination of labeling isotopes may be used, and it is not necessary to soak proteins out of gel slices.

Immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus: selection of the appropriate ligand and optimal conditions for elution.

A novel approach was used to select the most suitable antiviral monoclonal antibody (mAb) and elution conditions for immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus (MVE). Crude NS1 protein was subjected to a variety of chemical conditions produced by common elution buffers, and tested with a panel of NS1-specific mAbs by ELISA to determine which buffers denatured antigenic epitopes. Buffers that caused least structural damage to NS1 epitopes were tested by ELISA for dissociation of NS1-mAb complexes adsorbed to the solid phase. For each mAb analysed, the conditions required to break the mAb-NS1 complex on the solid phase were similar to those required to release antigen bound to mAb-sepharose beads. From these results we selected an appropriate antibody for affinity purification of the non-structural viral protein NS1 on CNBr-activated sepharose columns. Elution at pH 11.5 yielded good recoveries of highly pure and antigenically intact NS1 dimer. The results demonstrate that the appropriate ligand and optimal elution conditions can be rapidly determined generally by immunoassay in microtitre plates.

[Comparative analysis of the electrophoretic mobility of the high-molecular virus-specific proteins of flaviviruses]. / Sravnitel'nyi analiz élektroforeticheskoi podvizhnosti vysoko-molekuliarnykh virusspetsificheskikh belkov flavivirusov.

A comparative study of high molecular virus-specific proteins in cells infected with flaviviruses from 5 serological subgroups was carried out. Proteins NV5, NV4, and V3 were found to have similar electrophoretic mobility in viruses of the tick-borne encephalitis (TBE) complex with the exception of Powassan virus protein. Proteins NV5 and NV4 of mosquito-borne flaviviruses differ in electrophoretic mobility both from the corresponding proteins of the TBE complex viruses and from each other. Protein NV4 1/2 is formed only in cells infected with TBE complex viruses. Varying electrophoretic mobility of protein NV3 in different flaviviruses attests to its virus-specific nature.

[Heterogeneity of virus-specific flavivirus proteins]. / Geterogennost' virusspetsificheskikh belkov flavivirusov.

The polyacrylamide gel analysis of large intracellular virus-specific proteins NV5, NV4, and the intracellular form of structural protein V3 established differences in the electrophoretic mobility of each of these proteins formed in cells infected with tick-borne encephalitis, Powassan, Langat, and West Nile viruses. It is assumed that these differences in the electrophoretic mobility of NV5, NV4 proteins, and the intracellular form of V3 protein reflect the differences in the primary structure of each of these proteins in the viruses examined.

[Pathogenesis of persistent and chronic forms of tick-borne encephalitis (experimental study)]. / Patogenez persistentnoi i khronicheskoi form kleshchevogo éntsefalita (éksperimental'noe issledovanie).

A long-term experiment was conducted to study various aspects of the pathogenesis of persistent and chronic tick-borne encephalitis (TBE). Virological, serological, pathomorphological, electron microscopic and immunofluorescent techniques have been utilized in this study. Persistent TBE infection of Syrian hamsters examined over the period from 40 days to 2 years was characterized by the presence of virus-specific antigens in the organs and of specific antibodies in the blood serum. The persisting TBE virus was found to be predominantly localized in the central nervous system and spleen. Nerve cells underwent ultrastructural changes which were characteristic of flavivirus infection and related to the morphogenesis of viral particles. The authors have developed an experimental model of a primary progressive form of TBE with early and late manifestations of clinical symptoms of the disease.

Nomenclature of flavivirus-specified proteins.

Flavivirus structural proteins are designated E, C, and M, replacing the former nomenclature of V3 (envelope), V2 (core), and V1 (membrane-like) proteins, respectively. The nonstructural proteins, formerly NV1 through NV5, are designated by their apparent size in kilodaltons, prefixed by P (or GP if a glycoprotein), if they are stable unrelated end-products; the lower case p or gp is used for variants or uncharacterized products. A relationship to any structural protein is indicated by adding E, C, or M parenthetically to the designation of the appropriate nonstructural protein.

Molecular epidemiology of tick-borne encephalitis virus: peptide mapping of large non-structural proteins of European isolates and comparison with other flaviviruses.

Nine virus-specified proteins were identified by SDS-polyacrylamide gel electrophoresis in [35S]methionine-labelled chick embryo cells infected with tick-borne encephalitis (TBE) virus by comparison with mock-infected cells. These proteins were designated P91, p74, p72, P67, GP53(E), P47, p25, P15(C) and P14.5 according to their molecular weights. Peptide mapping of P91, P67, GP53(E) and P47 from TBE virus-infected cells, as well as those of the corresponding proteins from West Nile virus (WNV)-infected cells (previously termed NV5, NV4, V3 and NV3), demonstrated the uniqueness of these proteins. Almost no subtype variability, with respect to the pattern of intracellular proteins, was found when isolates of TBE virus from Austria, Switzerland, Germany, Finland and Czechoslovakia were compared. Peptide mapping of NV5 (P91) and NV4 (P67) from all these isolates using limited proteolysis with alpha-chymotrypsin and V8 protease revealed completely identical patterns, thus extending our observations that TBE virus seems to represent a very stable member of the flavivirus genus, which was based on the lack of variation found with the structural glycoprotein. On the other hand, the Far Eastern subtype of TBE virus and the closely related louping-ill virus could not only be differentiated from the Western subtype by differences in the peptide maps of their structural glycoprotein but also in those of the non-structural protein NV5, i.e. subtype or subgroup variations are not confined to the virion surface glycoprotein. WNV and Murray Valley encephalitis virus (MVEV) revealed the expected heterogeneity of virus-specified proteins found in cells infected with different flaviviruses. It is especially interesting that also the largest non-structural protein, NV5, is subject to this heterogeneity, ranging in mol. wt. from 91 000 for TBE virus to 98 000 for MVEV and that also the peptide maps of NV5, as well as those of NV4, were unrelated. These proteins, therefore, revealed a variability between serologically distinct flaviviruses similar to that observed with the structural glycoprotein.

Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses.

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

Peptide mapping of envelope-related glycoproteins specified by the flaviviruses Kunjin and West Nile.

Glycoproteins detected in Vero cells infected by the flaviviruses West Nile and Kunjin were examined by gel electrophoresis and peptide mapping. Two major glycoproteins, gp66 and gp54, were observed in West Nile virus-infected cells labelled for short time periods with [3H]mannose. A third glycoprotein, gp58, was present in smaller amounts. Pulse-labelling experiments suggested that gp66 was a precursor of gp54. Peptide mapping of [3H]leucine-labelled gp66, gp54 and the envelope glycoprotein E of West Nile virus demonstrated that gp66 and gp54 were related to E, and that the peptides of gp54 were a subset of those of gp66. Peptide mapping of the corresponding Kunjin virus-specified glycoproteins (gp66, gp59 and gp53) showed that the [3H]leucine-labelled peptides of gp53 and gp59 were similar and were contained within gp66. Since we have shown previously that gp59 and gp53 are related to E of Kunjin virus, we conclude that cells infected by West Nile or Kunjin viruses contain a similar set of E-related glycoproteins.

Characterization of novel viral polyproteins detected in cells infected by the flavivirus Kunjin and radiolabelled in the presence of the leucine analogue hydroxyleucine.

Vero cells were infected by Kunjin virus and radiolabelled in the presence of the leucine analogue, threo-beta-hydroxy-DL-leucine (THL). This analogue is known to prevent preprotein processing in cell-free systems when incorporated into signal peptides. Novel Kunjin virus-specified proteins were detected, namely gp140, p120 and gp92; the designations for the proteins indicate their approximate Mr X 10(-3) and whether they are glycosylated. The glycoproteins gp140 and gp92 were observed in cells labelled with [3H]mannose and bound to concanavalin A. Limited proteolytic digestion of gp140, gp92 and gp66 (related to the envelope protein E), and tryptic peptide mapping of these three glycoproteins and p120 indicated that all four were closely related. The glycoproteins gp140 and gp92 were also detected in cells infected by West Nile virus radiolabelled in the presence of THL. Other effects of THL in Kunjin virus-infected cells were a reduction in the incorporation of radioactive amino acids or mannose, a decrease in the yield of haemagglutinin and infectious virus, an inhibition of processing of gp66 to gp53 and an apparent inhibition of synthesis of P98 and P71. We suggest possible explanations for the THL-induced changes in infected cells based on recent models proposed for the synthesis of the yellow fever and West Nile viral proteins.

Positive identification of NS4A, the last of the hypothetical nonstructural proteins of flaviviruses.

A radiolabeled protein migrating in SDS-polyacrylamide gels near the core protein C of Kunjin virus-infected cells was isolated and subjected to N-terminal amino acid sequencing. Comparisons with the translation sequence deduced from the known nucleotide sequence identified a hydrophobic protein of 149 amino acids located in the polyprotein sequence between NS3 and NS4B, thus establishing its identity as NS4A with a calculated Mr 16,100. The cleavage sites identified at the N- and C-termini are KR decreases S....parallel....VAA decreases, both representing consensus sequences defined previously for Kunjin and other flaviviruses.

Envelope protein of the flavivirus Kunjin is apparently not glycosylated.

The envelope protein E (formerly designated V3) of the flavivirus Kunjin was not labelled with radioactive galactose, mannose or glucosamine during virus growth in Vero cells. On electrophoresis through polyacrylamide gels containing SDS, the envelope (E) protein migrated more rapidly than related intracellular virus-specified glycoproteins. Furthermore, E had a density in CsCl solution consistent with that of a protein lacking carbohydrate, and did not bind to concanavalin A-agarose. In contrast, the envelope glycoprotein of Murray Valley encephalitis virus (MVEV) did bind to concanavalin A under similar conditions and was readily labelled with radioactive mannose. These results suggested that the E protein of Kunjin virus was not glycosylated, a feature not shared with MVEV and West Nile virus (WNV), whose properties were consistent with the presence of oligosaccharides attached to the envelope proteins. When Kunjin virions were labelled with radioactive glucosamine, the label was contained in GP19 (formerly NV2). The glycopeptides derived by Pronase digestion of GP19 from Kunjin virions were larger than those derived from GP19 obtained from infected cells.

Carboxy-terminal analysis of nine proteins specified by the flavivirus Kunjin: evidence that only the intracellular core protein is truncated.

Nine proteins specified by Kunjin virus were labelled with [3H]lysine and digested with carboxypeptidase B which specifically cleaves carboxy-terminal Lys or Arg. The theoretical amount of [3H]lysine was released from the non-structural (ns) proteins NS2A, NS2B, NS3 and NS4B, which have a common carboxy terminus Lys-Arg deduced from cleavage sites established in the viral polyprotein by previous N-terminal amino acid analyses. This is a flavivirus consensus site, always followed by Gly, Ala or Ser. These results indicate that no truncations had occurred despite anomalous electrophoretic migrations of NS2A, NS2B and NS4B observed in some gel systems. No [3H]lysine was released from NS4A or NS5 which terminate in Ala and Leu, respectively, thus establishing that NS5 (observed Mr 98,000, theoretical Mr 103,600) was not cleaved post-translationally at any internal Lys-Arg site. Unexpectedly, [3H]lysine residues were apparently released from P14(C), the intracellular equivalent of virion C protein which terminates in Ala (adjacent to the established N-terminus of prM). However, a putative internal cleavage site (Lys-Arg decreases Gly) exists 18 residues upstream from the carboxy terminus of C, prior to the transmembrane spanning domain. Apparent internal cleavage in the cytosol at this site to produce P14(C) would expose [3H]lysine residues to carboxypeptidase B, and account for the previously observed differences in size and composition between C and P14(C).

Variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected Vero cells.

Indirect immunofluorescence with rabbit antisera was used to probe the intracellular locations of the antigens of envelope, prM (precursor to structural protein M) and the nonstructural glycoproteins NS 1 (formerly described as NV 3 or SCF) specified by the flaviviruses dengue-2 and Kunjin. Perinuclear staining in various types of foci was prominent for all antigens, and the distribution was influenced by whether cells were fixed with acetone or formaldehyde. Staining of Golgi-like masses or inclusions by anti-envelope sera occurred regularly and prominently in cells infected and stained with homologous anti-envelope antibodies; in the cross reactions, such staining was largely absent, especially in dengue-2 infected cells in which it was replaced by many small circular foci scattered throughout the cytoplasm. Anti-NS 1 also stained large perinuclear inclusions and small cytoplasmic foci, but the distribution of these was dissimilar to that observed with anti-envelope sera. Anti-prM appeared to contain a mixture of antibodies of different specificities, evident at different dilutions, possibly because of different cytoplasmic locations of prM and its cleavage products. All antisera produced small discontinuous foci on the plasma membrane of unfixed infected cells; antigens of NS 1 were sometimes prominent on the surface of acetone-fixed cells.

Comparisons by peptide mapping of proteins specified by Kunjin, West Nile and Murray Valley encephalitis viruses.

The relationships among virus-specified proteins of Murray Valley encephalitis (MVE), Kunjin (KUN) and West Nile (WN) viruses were investigated by peptide mapping of exhaustive proteolytic digests of radioactively labelled polypeptides. Maps of the three structural proteins (E, C and M) derived from purified virions and of two non-structural proteins (NV5 and NV4) obtained from infected cells were compared. For each polypeptide considered, the peptide maps of the KUN and WN virus-specified proteins were more similar to each other than either was to the map of the corresponding MVE virus-specified protein. Since the polypeptides considered together account for approximately 60% of the coding capacity of the flavivirus genome, our results suggested that, for the three viruses examined, the genomes of KUN and WN viruses are the most closely related.

Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: comparison with other flaviviruses.

Isolates of tick-borne encephalitis (TBE) virus from Finland, Germany, Czechoslovakia, Switzerland and Austria were compared with strains of the Far Eastern subtype isolated in Russia as well as Louping ill virus and other flaviviruses belonging to a different serocomplex: West Nile, Murray Valley encephalitis and Rocio viruses. Analysis of the structural polypeptides by SDS--polyacrylamide gel electrophoresis (SDS--PAGE) revealed identical mol. wt. of the glycoprotein E (mol. wt. 55 000) and the core protein C (mol. wt. 15 000) for all the TBE virus strains analysed. However, the small envelope protein M from viruses isolated in Germany, Switzerland and Austria migrated slightly slower (apparent mol. wt. 7500) compared to M from viruses isolated in Finland, Czechoslovakia or the Far Eastern subtype strains (apparent mol. wt. 6500 to 7000). The structural glycoproteins were isolated from purified [35S]methionine-labeled virions and subjected to peptide mapping by limited proteolysis with alpha-chymotrypsin or V8 protease followed by SDS--PAGE of the resulting cleavage products. With both proteases a remarkably homogeneous pattern was obtained for all the European isolates with only very minor deviations from a common pattern in single cases. Similar but distinguishable patterns were obtained for the Far Eastern subtype strains and also Louping ill virus, which, in addition, differed in the mol. wt. of its core protein C (mol. wt. 16 000) and the small membrane protein M (mol. wt. 9000). These almost identical peptide maps observed with the TBE virus strains were in sharp contrast to the unrelated patterns obtained with the glycoproteins from West Nile, Murray Valley encephalitis and Rocio viruses. Although these viruses are serologically closely related and members of the same serocomplex of flaviviruses their glycoprotein peptide maps were completely different from one another. In a competitive radioimmunoassay all European TBE virus isolates showed identical immunological reactivity which further points to the great stability of this type of virus.

Acidotropic amines inhibit proteolytic processing of flavivirus prM protein.

Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weight, glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM occurs in the acidic post-Golgi vesicles.

Separation of flavivirus membrane and capsid proteins by multistep high-performance liquid chromatography optimized by immunological monitoring.

Complete separation of the three structural proteins, E, C and M, of an enveloped virus (tick-borne encephalitis virus) was achieved by means of a two-step high-performance liquid chromatography (HPLC) technique in less than 1 h. The hydrophobically associated membrane proteins E and M were successfully separated by high-performance gel permeation chromatography (TSK-3000 SW column) in the presence of sodium dodecyl sulphate (SDS), whereas the separation of M and C as well as desalting and removal of SDS was achieved by subsequent reversed-phase chromatography on a C3 column. With regard to further characterization by peptide mapping, analysis of the amino acid composition and aminoterminal sequencing, the second step was performed with volatile buffer systems. Quality control of the separation was achieved by a combination of HPLC with a highly sensitive dot immunoassay by the use of polyclonal as well as monoclonal antibodies. This method proved extremely sensitive and revealed strong tailing effects and cross-contaminations of peaks well-separated in reversed-phase chromatography, which were neither apparent in the absorbance curve at 214 nm nor in the analysis by SDS-polyacrylamide gel electrophoresis. By visualization of the peak-tailing effect, the chromatographic conditions could be modified in order to achieve an optimum separation of proteins.

The relationship between the flaviviruses Skalica and Langat as revealed by monoclonal antibodies, peptide mapping and RNA sequence analysis.

The flavivirus Skalica was isolated from a bank vole in Czechoslovakia in 1976. It can be serologically distinguished from prototype strains of tick-borne encephalitis (TBE) virus and has a decreased virulence for adult mice. We have further defined the relationship of Skalica virus to other members of the TBE serocomplex (TBE European and Far Eastern subtypes, Langat and louping ill virus) by using a panel of 22 monoclonal antibodies, peptide mapping and RNA sequence analyses. By these criteria Skalica virus proved to be distinct from TBE virus and to be very closely related to Langat virus, differing by only two bases among a total of 416 nucleotides compared. The sequence of 22% of the Langat genome was determined and the encoded amino acid sequences were derived. Comparison of these with the corresponding amino acid sequences of TBE virus revealed a similarity of 85%, as opposed to 93% similarity between the European and Far Eastern subtypes of TBE virus.