Characterization of Protease Cleavage Sites Involved in the Formation of the Envelope Glycoprotein and Three Non-structural Proteins of Dengue Virus Type 2, New Guinea C Strain

Abstract

SUMMARYAmino 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-GlnArg-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.

Comparative immunological recognition of proteins from New Guinea "C" dengue virus type 2 prototype and from a dengue virus type 2 strain isolated in the State of Rio de Janeiro, Brazil

“…We observed that the protein molecular mass is not exactly the same as described by other authors (Se- Thoe et al 1999). In this context, it is important to have in mind that the heterogeneity in the molecular mass of those proteins could be explained by both viral and host proteases that may be involved in processing the flavivirus proteins (Biedryzcka et al 1987). Moreover, it is known that some flavivirus proteins reach different migration levels in SDS gels considering the intracellular and virion-associated forms (Wright & Westaway 1977, Svitkin et al 1984.…”

Gene Mapping and Positive Identification of the Non-structural Proteins NS2A, NS2B, NS3, NS4B and NS5 of the Flavivirus Kunjin and Their Cleavage Sites

“…viruses (Rice et al, 1985(Rice et al, , 1986aBiedrzycka et al, 1987). The matched nucleotide and amino acid sequences for KUN virus at the cleavage sites are shown in Fig.…”

“…Where the amino acid residue in position -3 is not a short side chain amino acid, a cluster of three basic amino acids occurs. Sources of sequence data are as follows: WN virus (Castle et al, 1986), MVE virus (Dalgarno et aL, 1986), JE virus (Sumiyoshi et aL, 1986), SLE virus (Trent et al, 1987), YF virus (Rice et al, 1985) and DEN-2 virus (Biedrzycka et aL, 1987;P J. Wright, personal communication) endoplasmic reticulum (ER). Hence KUN NS 1 appears likely to be cleaved by signal peptidase.…”

In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids

“…In contrast, the amino termini of proteins NS2B, NS3, NS4A and NS5 are preceded by a protease consensus sequence which contains multiple basic amino acid residues (Rice et al, 1985(Rice et al, , 1986Biedrzycka et al, 1987;Speight et al, 1988;Chambers et al, 1989;Wright et al, 1989;Wengler et al, 1990) and it has been proposed that a cytosolic virus-encoded protease which cleaves after double basic residues is responsible for the corresponding proteolytic cleavages (Rice et al, 1985;Speight et al, 1988). Recently, a computer search of conserved amino acid residues has led to the suggestion that the amino-terminal segment of the NS3 protein represents a serine protease (Bazan & Fletterick, 1989;Gorbalenya et al, 1989).…”