title: Mutagenesis of conserved residues at the yellow fever virus 3/4A and 4B/5 dibasic cleavage sites: effects on cleavage efficiency and polyprotein processing.
abstract: Flavivirus proteins are produced by co- and post-translational proteolytic processing of a large polyprotein using both host- and virus-encoded enzymes. The flavivirus serine proteinase, which consists of NS2B and NS3, is responsible for cleavages of at least four dibasic sites in the nonstructural region. In this study, a number of substitutions for the conserved amino acids flanking the 3/4A and 4B/5 dibasic cleavage sites [Arg(P2)-Arg(P1) decreases Gly(P1')] were examined for their effects on yellow fever virus (YF) polyprotein processing. The substrate for these studies was a truncated YF polyprotein, called sig2A-5(356), which consists of a signal sequence fused to NS2A and extending through the first 356 amino acids of NS5. At the P1' position (Gly) of the 4B/5 site, only Ser and Ala were allowed while six other substitutions abolished cleavage. Substitutions of the 4B/5 P1 Arg residue with Lys, Gln, Asn, or His were tolerated while replacement with Glu eliminated cleavage. The 4B/5 P2 position (Arg) was found to be tolerant of substitutions with polar or hydrophobic residues which allowed varying degrees of partial cleavage. Previous studies have shown that cleavage at the 3/4A site is incomplete in YF-infected cells and that the cleavage efficiency at this site is significantly less for the sig2A-5(356) polyprotein. Replacement of the 3/4A P1 Arg residue with noncharged polar or hydrophobic residues reduced the cleavage efficiency, whereas substitutions with Glu or Pro abolished cleavage. Studies with polyproteins containing one or both of the 3/4A and 4B/5 cleavage sites blocked indicate that there is not an obligatory processing order for cleavages generating the N termini of YF NS4A, NS4B, and NS5.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
authors.forename: C
authors.initials: C
authors.lastname: Lin
authors.forename: T J
authors.initials: TJ
authors.lastname: Chambers
authors.forename: C M
authors.initials: CM
authors.lastname: Rice
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015005:Yellow fever virus / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D017361:Viral Nonstructural Proteins; D012697:Serine Endopeptidases