The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase.

Recently it has been reported that a membrane fraction can be isolated from West Nile virus-infected BHK cells which contains the viral nonstructural (NS) proteins as major constituents (Wengler et al., 1990). In this report we show that treatment of these membranes with subtilisin releases the carboxy-terminal segment of the NS 3 protein as a soluble protein of about 50 kDa apparent molecular weight. This molecule, which is called the p50-S protein, can be purified by standard chromatographic procedures. The p50-S protein binds to poly(A) and apparently represents a nucleoside triphosphatase which is stimulated in the presence of ssRNA molecules. The data represent experimental support for the predicted role of this segment of the NS 3 protein as an RNA helicase. Some properties of the p50-S protein are described and a possible function of this protein segment during RNA synthesis is discussed.

Comparative analysis of West Nile virus strains isolated from human and animal hosts using monoclonal antibodies and cDNA restriction digest profiles.

Three West Nile (WN) virus strains isolated in Bangui, Central African Republic (CAR), from patients with hepatitis were analysed comparatively with the prototype WN virus strain and 7 WN strains previously isolated from birds (2 strains), mosquitoes (3 strains) and ticks (2 strains) in CAR. The comparison was based on two techniques: an epitopic analysis by indirect immunofluorescence assay using a panel of 9 monoclonal antibodies to WN virus, and an analysis of HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA. Similar results were obtained with both techniques: the 3 human strains were found to be identical to each other and identical or very close to mosquito and tick strains, whereas prototype WN virus and bird strains were significantly different from the human strains. As "classical" infections due to WN virus without hepatic involvement were also reported during the period of isolation of the arthropod strains, we concluded that the same virus subtype may have been the cause of different infection patterns. A new definition of the disease spectrum of WN virus, including the possibility of liver involvement, should be established. Clearly, the Egyptian prototype WN virus represents a different topotype. Bird strains also appear to be different from human and arthropod strains, raising the question of their transmissibility and pathogenicity for man, and of the role of birds in the natural cycle of WN virus.

West Nile virions aligned along myelin lamellae in organotypic spinal cord cultures.

Organotypic spinal cord cultures infected with West Nile Virus (WNV) exhibited a remarkable arrangement of virions among lamellae of the myelin sheath. Virions were first observed in neurons and only at day 4 after infection appeared within the myelin lamellae. Virions were observed only in the central myelin, aligned along the interperiod lines and therefore attached to the outer side of the oligodendrocyte membrane. In spite of this peculiar location of the virions, their presence was not associated with severe damage to the axon or to the myelin sheath. The causes of the formation of this viral pattern, its morphological features, and the role which it might have in viral infection in vivo are considered.

Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis.

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre-M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues 1-105, 124-290, and 291-787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells.

Analysis of the influence of proteolytic cleavage on the structural organization of the surface of the West Nile flavivirus leads to the isolation of a protease-resistant E protein oligomer from the viral surface.

In order to analyze the organization of the membrane proteins pre M, M, and E of the West Nile (WN) flavivirus we have studied the influence of proteolytic cleavage of intact virus on the structure of these proteins. The amino acid sequence of all proteins is known, all six disulfides present in the viral E protein have been identified, and it has been suggested that the E protein contains regions R1, L1, R2, L2, and R3, which together form the E protein ectodomain followed by a carboxyterminal membrane anchor region (Th. Nowak and G. Wengler (1987) Virology 156, 127-137). The results of our analyses can be summarized as follows: (1) The surface of the WN virus contains E protein oligomers; the E protein molecules present in these structures contain two segments which are exposed to proteolytic attack; the segments are located in parts L1 and R3 of the E protein. (2) Proteolytic cleavage of these oligomers in these regions neither destroys nor releases the oligomers from the viral surface. (3) The WN virus surface contains a layer of 7-nm ring-shaped subunits identifiable by electron microscopy which are neither destroyed nor released by proteolytic cleavage. (4) An E protein trimer can be isolated from the surface of protease-treated WN virus. This trimer is morphologically similar to the 7-nm ring-shaped element which can be identified on the surface of native and protease-treated WN virus by electron microscopy.

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.

Analysis of disulfides present in the membrane proteins of the West Nile flavivirus.

Recently the primary structure of the structural proteins of the flaviviruses West Nile (WN) virus (Castle et al., 1985; Wengler et al., 1985) and yellow fever (YF) virus (Rice et al., 1985) have been determined. As a first step in a further characterization of the organization of the structural proteins we have now studied the disulfide bridges present in the WN virus membrane proteins. All three membrane proteins, pre M, M, and E, were analyzed. The results obtained can be summarized as follows: The pre M proteins of both WN and YF virus each contain 6 cysteine residues and the position of all of these residues is strictly conserved between both viruses. The M proteins of both viruses do not contain cysteine residues. The E proteins of these viruses contain 12 cysteines and the position of all of these residues is strictly conserved between both viruses. All cysteine residues of the WN virus-derived membrane proteins are present as intramolecular disulfides. The six disulfide bridges generated from the 12 cysteine residues in the WN virus-derived E protein have been identified as follows: Cys 1-Cys 2; Cys 3-Cys 8; Cys 4-Cys 6; Cys 5-Cys 7; Cys 9-Cys 10; Cys 11-Cys 12. The analyses of the amino acid sequence conservation between the E proteins of YF and WN virus and the characterization of the disulfides have been used to develop a description of the E protein in which the molecule is assumed to be composed of the segments R1, L1, R2, L2, and R3 followed by a membrane anchor region at the carboxy-terminal region of the molecule. Computer analyses of the hydrophilicity and of the secondary structure indicate that the R1 region might contain a cluster of viral epitopes.

Sequence analysis of the membrane protein V3 of the flavivirus West Nile virus and of its gene.

Flaviviruses contain a large membrane-associated protein V3, having a mol mass of about 50 kDa which is responsible for hemagglutination. We have isolated the V3 protein from the West Nile (WN) flavivirus and determined its amino-terminal amino acid sequence and amino acid sequences of fragments derived from this protein. We have also transcribed parts of the WN virus genome RNA into cDNA and cloned and sequenced this cDNA. The results of these analyses have allowed us to identify the region of the viral genome coding for the V3 protein. In this report we describe the total nucleotide sequence of the genome region coding for the WN virus V3 protein and the amino acid sequence of the V3 protein derived from these analyses. The exact carboxy terminus of the V3 protein has not been determined in these experiments. These analyses have shown that the V3 protein of WN virus does not contain an Asn-X-Ser/Thr sequence which could allow addition of N-linked carbohydrate chains to this protein. In accordance with this finding, analyses of metabolic labeling of the V3 protein using [3H]glucosamine indicate that the WN virus V3 protein is an unglycosylated protein. Together with our earlier analyses these results show that the viral structural proteins are present on the genome RNA in the order 5'-terminus-core protein (V2)-small membrane-associated protein (NV2)-large membrane-associated protein (V3) and describe the nucleotide sequences coding for all WN virus structural proteins identified so far. A hypothesis concerning the processes involved in the synthesis of all viral structural proteins and the probable orientation of these proteins relative to the endoplasmatic reticulum membrane based on the structure of these proteins is discussed.

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.

Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures.

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.

[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.

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.

Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.