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To gain insight into the role of cell surface glycosaminoglycans (GAG) in dengue virus (DEN) cell tropism and virulence, DEN-2 mouse brain-adapted vaccine candidate, neurovirulent prototype strain (NGC) and low-passage strain, PUO-218, were passaged in BHK-21 and SW13 cells to isolate variants with high affinity for GAG. Sequence comparisons of parent and passage variants revealed five GAG-binding determinants, which all cluster in a surface-exposed region in domain II of the three-dimensional structure of the DEN envelope protein. Using an infectious cDNA clone of NGC and an NGC/PUO-218 prM-E chimeric clone, it was demonstrated that the GAG-binding determinants augment the specific infectivity for BHK-21 and/or SW13 cells by 10- to 170-fold and in some cases marginally reduce that for Vero cells. This altered cell tropism was due to a greater dependence of the variants on cell surface GAG for attachment/entry, given their increased susceptibility to heparin inhibition. The effect of the GAG-binding determinants on virulence was examined in mice deficient in alpha/beta/gamma interferon responses. High GAG affinity strongly correlated with low neuroinvasiveness due to rapid virus clearance from the blood. It was speculated that this mechanism accounts for the attenuation in primates of some DEN vaccine candidates. Interestingly, the GAG-binding variants did not display marked attenuation of neurovirulence and the opposing effect of enhanced neurovirulence was associated with one determinant (Lys126) already present in mouse brain-adapted NGC. This discrepancy of attenuated neuroinvasiveness and augmented neurovirulence may be reconciled by the existence of different mechanisms of virus dissemination in the brain and in extraneural tissues.

The mature flavivirus particle comprises a nucleocapsid core surrounded by a lipid bilayer containing the membrane (M) (derived from the precursor prM) and envelope (E) proteins. The formation of intracellular prM/E heterodimers occurs rapidly after translation and is believed to be important for the assembly and secretion of immature virus particles. In this study, the role of the His residue at position 39 in the M protein (M39) of dengue virus type 2 (DENV-2) in the virus life cycle was investigated. Mutations encoding basic (Arg), non-polar (Leu and Pro) and uncharged polar (Asn, Gln and Tyr) amino acids at M39 were introduced into a DENV-2 genomic-length cDNA clone and their effects on virus replication were examined. Substitution of the His residue with non-polar amino acids abolished virus replication, whereas substitution with basic or uncharged polar amino acids decreased virus replication moderately ( approximately 2 log(10) p.f.u. ml(-1) decrease in viral titre for Arg and Asn) or severely (>3.5 log(10) p.f.u. ml(-1) decrease in viral titre for Gln and Tyr). Selected mutations were introduced into a prM-E gene cassette and expressed transiently in COS cells to investigate whether the mutations impaired prM/E association or secretion. None of the mutations was found to disrupt the formation of intracellular prM/E heterodimers. However, the mutations that abolished virus replication prevented secretion of prM/E complexes. The results of this study pinpoint a critical residue in the M protein that potentially plays a role in viral morphogenesis, secretion and entry.

The N-terminal one-third of the NS3 protein of Dengue virus type 2 (DEN-2) complexes with co-factor NS2B to form an active serine proteinase which cleaves the viral polyprotein. To identify sites within NS3 that may interact with NS2B, seven regions within the NS3 proteinase outside the conserved flavivirus enzyme motifs were mutated by alanine replacement. Five sites contained clusters of charged residues and were hydrophilic. Two sites were hydrophobic and highly conserved among flaviviruses. The effects of five mutations on NS2B/3 processing were examined using a COS cell expression system. Four retained significant proteinase activity. Three of these mutations and two more were introduced into genomic-length cDNA and tested for their effects on virus replication. The five mutant viruses showed reduced plaque size and two of the five showed significantly reduced titres. All seven mutations were mapped on the X-ray crystal structure of the DEN-2 NS3 proteinase: three were located at the N terminus and two at the C terminus of the NS2B-binding cleft. Two mutations were at the C terminus of the proteinase domain and one was solvent-exposed. The study demonstrated that charged-to-alanine mutagenesis in the viral proteinase can be used to produce growth-restricted flaviviruses that may be useful in the production of attenuated vaccine strains.

The three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the basis of earlier and current results with transient expression, six mutations which reduced NS1 dimerization and two mutations which inhibited prM/E cleavage were analysed individually for their effects on virus growth using a genomic length cDNA clone. Two viruses were obtained that showed reduced growth in cell culture and attenuation of neurovirulence when inoculated into 3-day-old mice. One of these viruses encoded NS1 that lacked the second glycosylation site, the other encoded a Ser --> Ile change at the -3 position of the prM/E cleavage site. A third virus encoding a mutation in NS1 within a hydrophilic region grew as well as the parental virus. No virus was detected for the remaining five mutations.

A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coil and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus (MON310) that replicated well in C6/36 cells but was not neurovirulent in mice. MON310 contained the prM and E genes of the non-neurovirulent PUO-218 strain in an NGC background. There were seven amino acid differences between the prM and E proteins of MON310 and MON501. The differences were generally conservative, with the exception of E residue 126, which was Glu in MON310 and Lys in MON501. To examine the role of this residue in mouse neurovirulence, substitutions of Glu --> Lys and Lys --> Glu were made in MON310 and MON501, respectively. The properties of these mutants clearly demonstrated that Lys at E residue 126 is a major determinant of DEN-2 mouse neurovirulence.

The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid. The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460... within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.

Sequencing of the S1 genes of nine Australian strains of infectious bronchitis virus (IBV) identified two genotypically distinct groups of strains. The strains Vic S, V5/90, N1/62, N3/62, N9/74, and N2/75 comprised group I, sharing 80.7-98.3% identity in their deduced amino acid sequences. All group I strains were able to replicate in the trachea and kidney but only four strains, Vic S, N1/62, N9/74, and N2/75, were nephropathogenic, the latter three causing mortalities ranging from 32 to 96%. Group II contained strains N1/88, Q3/88 and V18/91 which only replicated in the trachea, inducing no mortalities. These viruses showed 72.3-92.8% amino acid identity to each other and only 53.8-61.7% identity to viruses of the first group. They were also distinct from the Massachusetts 41 and D1466 strains (47.5-55.7% amino acid identity). Thus N1/88, Q3/88 and V18/91 form a new group of viruses which are genotypically distinct from all previously characterized IBV strains. No definite correlations were established between the S1 amino acid sequences and the nephropathogenicity of strains.

The non-structural glycoprotein NS1 of dengue virus type 2 contains sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 synthesized in infected cells is glycosylated at both locations. We have now examined the dimerization and secretion of NS1 lacking one or both of these sites by transient expression of mutagenized cDNA inserted into a simian virus 40-based vector. Immunoblotting and radioimmunoprecipitation were used to detect NS1 associated with transfected cells and in the extracellular medium. Elimination of one or both glycosylation sites did not abolish dimerization and secretion of NS1. However, NS1 lacking Asn-207 showed reduced dimer stability and secretion. Treatment of secreted NS1 with endoglycosidase H demonstrated that complex glycans were attached at Asn-130 and high-mannose glycans at Asn-207.

A glycoprotein C-prM of 35,000 M(r) was immuno-precipitated from lysates of Aedes albopictus cells infected with dengue virus type 2 (DEN-2) using antisera directed against the C protein or an amino-terminal fragment of the prM glycoprotein. C-prM was not detected in infected Vero cells. The prM glycoprotein synthesized in infected A. albopictus and Vero cells was cleaved to produce the membrane-associated virion protein (M) and the non-M fragment (pr) immediately preceding or occurring simultaneously with the release of viral particles from cells. The cleavage was less efficient in mosquito cells. The pr fragment was found only in the medium and was not rapidly degraded. To obtain pr-specific and M-specific antisera for these studies, proteins containing fragments of DEN-2 prM fused with staphylococcal Protein A were synthesized in Escherichia coli using the expression vector pRIT2T. The fusion proteins were stable and were used to raise antisera in rabbits for immunoprecipitation of radiolabelled cell extracts and culture medium. This is the first report of the detection of a C-prM protein in flavivirus-infected cells and the identification of the pr component of prM.

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.

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.

Fifteen proteins were detected in Vero cells infected by dengue type 2 (DEN-2) virus that were not observed in mock-infected cells, namely P98, p82, P67, GP60, gp54, GP46, p30, p28, gp22, GP20, p18, gp16, p15, p14 and gp13. With the exceptions of gp54 and gp13, polypeptides corresponding to those listed above were also observed in DEN-2 virus-infected Aedes albopictus C6/36 cells. Pulse-chase labelling experiments suggested a possible precursor-product relationship between p30 and p28, and between gp22 and GP20. Peptide mapping and immunoprecipitation experiments showed that the major glycoproteins GP60, GP46 and GP20 were unrelated. Immunoprecipitations of infected cells with antiserum prepared against the DEN-2 soluble complement-fixing (SCF) antigen demonstrated that this antigen is equivalent to the non-structural glycoprotein GP46. The envelope glycoprotein (E) from virus grown in C6/36 cells migrated faster through polyacrylamide gels containing SDS than E from virus grown in Vero cells. [3H]Mannose-labelled glycopeptides of GP60, GP46 and GP20 were separated by gel filtration and by electrophoresis in Tris-borate gels; in addition, the polypeptides synthesized in infected cells in the presence of tunicamycin were analysed. The results revealed heterogeneity among the glycan units of GP60 and GP46.

Amino terminal sequences of the envelope protein E and the three largest nonstructural proteins NS1, NS3, and NS5 of the New Guinea C strain of dengue virus type 2 (DEN-2) were obtained by nucleotide and protein sequencing. Clones were prepared containing cDNA of DEN-2 virus in the plasmid pUC8. The nucleotide sequences of viral cDNA inserts were determined and the cDNA of each clone positioned on the flavivirus genomic map by comparison of the deduced amino acid sequence with that of yellow fever virus. Radiolabelled E, NS1, NS3 and NS5 were purified by lectin affinity chromatography and preparative gel electrophoresis. Purified proteins were subsequently analysed by Edman degradation to establish the origins of the amino termini of these proteins in the deduced DEN-2 amino acid sequence. Thus the amino acid sequences surrounding the likely proteolytic cleavage sites used in the formation of these four proteins were determined. Of particular interest was the sequence containing the amino terminus of NS3, namely Lys-Lys-Gln-Arg-Ala-Gly where Ala is the first amino acid of NS3. Cleavage following one basic residue in the flavivirus polyprotein has not been reported previously.

The proportion of cells absorbing trypan blue (tb-+ character) can be used to measure the late c.p.e. of wild-type poliovirus (ts-+. tb-+), which was the same at restrictive (39-2 to 39-6 degrees C) or permissive (37 degrees C) temperatures. Of twenty ts mutants, seven showed normal c.p.e. at 37 degrees C but were defective in C.P.E. (TB) AT 39-5 degrees C; all seven tb mutants have previously been shown (Cooper et al. 1971) to give evidence of a primary defect in replicase 1 activity (to make the complementary or minus strand of virus RNA). The remainder (tb-+) have all previously been shown to give evidence of a primary defect either in replicase II activity (to make progeny plus strands) or in structural protein. Thus, the late c.p.e. is dependent on a product of the replicase I gene, of which the in vivo effector is probably double-stranded RNA. Late c.p.e. is not caused by prevention of host protein, RNA or DNA synthesis and is not necessarily correlated with lysosomal enzyme release. The tb mutants were also defective in inducing early changes in chromatin (chr) and in prevention of thymidine incorporation (pti), but the tb and pti/chr characters are probably independent expressions of replicase I activity. Virus growth does not depend on repression of DNA synthesis. Poliovirus represses the activities of host DNA-dependent RNA polymerase I and II to an equal extent. There is no evidence that repression of DNA or RNA synthesis results from direct interaction of virus protein with the DNA.

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.

The nucleotide sequence and the deduced amino acid sequence for the genes encoding the structural proteins of two strains of dengue virus type 2 (DEN-2) were determined from cDNA clones. The genes for C, prM(M) and E proteins were sequenced for the prototype DEN-2 virus, the New Guinea C strain. Also sequenced were the prM(M) and E genes of PUO-218. This strain of DEN-2 was isolated during 1980 in Bangkok and had received a limited number of laboratory passages. Comparisons of the newly determined sequences with those published for the Jamaica 1409 and Puerto Rico PR-159 (S1 vaccine candidate) strains revealed a close relationship between New Guinea C virus and both the Jamaica and PUO-218 viruses (greater than 96% similarity in nucleotides of the E gene), whereas S1 virus was the most divergent.

Viral polypeptides associated with the membrane-free replication complex of poliovirus RNA were multiple in nature. The structural protein precursors [VP0, VP1, VP3] predominated, and because they were found in a cytoplasmic component with the same S value and density as the replication complex are likely to be attached to it in vivo. They were not present in the form of empty capsids. The electrophoretic polypeptide pattern of the membrane-bound replication complex was similar but showed a predominance of NCVPX or VP1, unless the cells were slightly depleted in amino acids when the non-structural polypeptide NCVP2 became important. Cystine was the only amino acid capable of reversing this depletion effect on its own.