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.
A virus-encoded protease that cleaves after multiple basic amino acid residues has been implicated in the processing of the flavivirus polyprotein. Recently, a computer search of amino acid residues which might form the active site of a protease led to the suggestion that the amino-terminal segment of the NS3 protein represents a serine protease. To examine this possibility we constructed an mRNA which encodes a polyprotein with an amino-terminal signal sequence derived from the influenza virus haemagglutinin, followed by a segment of the West Nile flavivirus polyprotein which includes the non-structural (NS) proteins NS2A, NS2B and the amino-terminal part of the NS3 protein. This polyprotein contains two sequences, located at the termini of the NS2B protein, which are cleaved by the viral protease that cleaves after multiple basic residues in the authentic polyprotein. The proteins that are generated by this mRNA during in vitro translation in the presence of rough endoplasmic reticulum membranes indicate that these two proteolytic cleavages occur in vitro. In vitro translation of polyproteins shortened at the carboxy terminus shows that a polyprotein which does not contain the complete set of proposed catalytic residues present in the NS3 protein segment accumulates as a membrane-associated molecule without proteolytic processing. Similarly, substitution of residue histidine 51 of the NS3 polyprotein segment, which is predicted to be part of the protease catalytic centre, with an alanine residue, blocks the processing of the polyprotein in vitro.
Wengler
G
G
Institut für Virologie, Justus-Liebig-Universität Giessen, Germany.
Czaya
G
G
Färber
P M
PM
Hegemann
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005814:Genes, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*; D014902:West Nile virus / Q000235:genetics* / Q000378:metabolism
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D010450:Endopeptidases