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

Abstract

SUMMARYPartial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44-P19.P10.P71.(?)-P21-P98-3', YF 5'...NSl.ns2a.ns2b.NS3.ns4a-ns4b.NS5-3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg~Gly, similar in form to the sequence Arg-Arg~Ser defined at the cleavage sites ofYF NS3 and NSS. A new consensus cleavage site for NS1, NS2A and NS4B in the form VaI-X-Ala~, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flav ivirus sequences (a total of 18 sites). N S 1 and N S4B, but not N S2A, were preceded by a putative signal sequence.

Virus-encoded proteinases of the Flaviviridae.

“…Experiments on a number of different flaviviruses showed that the cleavages between the C\prM, prM\E, E\NS1, NS1\NS2A and NS4A-B proteins are mediated by host-cell proteinases (Biedrzycka et al, 1987 ;Nowak et al, 1989 ;Ruiz-Linares et al, 1989 ;Speight et al, 1988 ;Markoff, 1989 ;Wright et al, 1989 ;Lin et al, 1993 ;Lobigs, 1993 ;Yamshchikov & Compans, 1994Falgout & Markoff, 1995). Sites of other processing events (identified by N-terminal sequencing of virus proteins) occurred at motifs commonly consisting of dibasic amino acid pairs at P1 and P2 followed by a small, non-branched amino acid at P1h (Rice et al, 1986 ;Biedrzycka et al, 1987 ;Speight et al, 1988 ;Chambers et al, 1989). These sites proved to be conserved amongst the flavivirus sequences then becoming available (Rice et al, 1985 ;Castle et al, 1986 ;Sumiyoshi et al, 1987 ;Coia et al, 1988 ;Hahn et al, 1988 ;Pletnev et al, 1990).…”

“…Sites of other processing events (identified by N-terminal sequencing of virus proteins) occurred at motifs commonly consisting of dibasic amino acid pairs at P1 and P2 followed by a small, non-branched amino acid at P1h (Rice et al, 1986 ;Biedrzycka et al, 1987 ;Speight et al, 1988 ;Chambers et al, 1989). These sites proved to be conserved amongst the flavivirus sequences then becoming available (Rice et al, 1985 ;Castle et al, 1986 ;Sumiyoshi et al, 1987 ;Coia et al, 1988 ;Hahn et al, 1988 ;Pletnev et al, 1990). Recently, it has been shown that a cleavage within the final helicase motif in the Cterminal region of dengue virus type 2 NS3 (Fig.…”

“…All C termini are arbitrary, but reflect an appropriate region where the alignment is maintained across all the groups of viruses before the similarity drops too low. Sequences used in the analyses : dengue virus (type 1 - Fu et al, 1992 ;type 2 -Hahn et al, 1988 ;type 3 -Osatomi & Sumiyoshi, 1990 ;type 4 -Mackow et al, 1987) ; Japanese encephalitis virus (Sumiyoshi et al, 1987) ; Kunjin virus (Coia et al, 1988) ; Murray Valley encephalitis virus (MVEV -Dalgarno et al, 1986) ; tick-borne encephalitis virus (TBEV -Pletnev et al, 1990) ; West Nile virus (WNV Castle et al, 1986) ; yellow fever virus (YVF - Rice et al, 1985) ; hepatitis G virus (strain GB-A, B - Simons et al, 1995 ;C -Leary et al, 1995) ; hepatitis C virus (HCV strain 1a -Choo et al, 1991 ;1b -Kato et al, 1990 ;1c -Okamoto et al, 1994 ;2a -Okamoto et al, 1991 ;2b -Okamoto et al, 1992 ;2c -Nakao et al, 1996 ;3a -Sakamoto et al, 1994 ;3b -Chayama et al, 1994 ;4a -Chamberlain et al, 1997 a ;5a -Chamberlain et al, 1997 b ;6a -Adams et al, 1997) ; bovine viral diarrhoea virus (BVDV strain Osloss - Renard et al, 1987 ;De Moerlooze et al, 1993 ;NADL -Collet et al, 1988 b ;SD1 -Deng & Brock, 1992 ;NCP7 -Meyers et al, 1996 ;ILLC -Roath & Berry, unpublished ;IIRidpath & Bolin, 1995) ; classical swine fever virus (CSFV strain Alfort -Meyers et al, 1989 ;Brescia -Moorman et al, 1990 . Sequence similarities between NS3 proteolytic domains of the Flaviviridae.…”

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).…”

“…All virus-encoded proteins identified thus far are derived from the encoded polyprotein. Analysis of terminal sequences of virus-encoded proteins has shown that the viral structural and non-structural (NS) proteins are organized on the polyprotein in the order, core protein-envelope protein (pre-M)-envelope protein (E) -NS1 -NS2A -NS2B -NS3 -NS4A -NS4B -NS5 (Rice et al, 1985(Rice et al, , 1986Castle et al, 1986;Speight et al, 1988;Chambers et al, 1989;Speight & Westaway, 1989b;Wright et al, 1989;Wengler et al, 1990).…”