Summary
A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5′-C·GP20·E·GP44·P19·P10·P71·(?)·P21·P98-3′ YF 5′-C·prM·E·NS1·ns2a·ns2b·NS3·ns4a·ns4b·NS5-3′. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala↓ where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg↓Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN > Murray Valley > Japanese encephalitis > St Louis encephalitis viruses within this closely related serological complex. A non-coding 5′ sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3′ sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins.

Publisher Summary The family Flaviviridae comprises 65 species, which globally represent the most widespread and medically important of the arthropod-borne viruses. Knowledge of flavivirus replication strategy has accumulated slowly—current information indicates that the strategy is apparently simple, involving the genome as the single capped messenger with one open reading frame of 10,233 nucleotides, and no involvement of the cell nucleus. Among the unusual features of flavivirus replication awaiting elucidation are the long latent period, the variable production and the function of small ns proteins, the anomalous translation of the most 3' distal and largest product NV5 in translation mapping experiments, the inconsistencies in carbohydrate content of envelope protein, and the apparent absence of nucleocapsids and budding in virus morphogenesis. Some of these problems may be solved as more amino acid and nucleotide sequence data are acquired. This information may lead to the development of genetically engineered and safe flavivirus vaccines or may provide a rationale for antiviral chemotherapy. Meanwhile, the events programmed by the flavivirus genome—the largest messenger fully expressed in eukaryotic cells—and the responses of host cells may continue to intrigue and challenge the growing band of flavivirus researchers.

Flavivirus Replication Strategy

The flaviviruses were formerly classified as a genus in the family Togaviridae. They have now been elevated to family status, family Flaviviridae, in part because of differences in replication and assembly (Westaway, 1980) (Chapter 11) and in part because their genome structure is quite different from that of the alphaviruses (Rice et al., 1985) (compare with Chapter 3). With the determination of the complete nucleotide sequence of the yellow fever virus genome and a large portion of the Murray Valley encephalitis virus genome, it has become clear that these viruses represent a distinct group among the plus-stranded RNA viruses. This chapter will focus on the implications of these and other recent sequence data on flavivirus gene expression, replication, and evolution.

Structure of the Flavivirus Genome

Partial nucleotide sequence of the Murray Valley encephalitis virus genome. Comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins.

Summary
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.

Marilyn Shew

Papers

Proteins and glycoproteins specified by the flavivirus Kunjin