title: Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein.
abstract: The yellow fever virus NS2B-3 proteinase mediates cleavages within the nonstructural region at a consensus sequence defined by G/ARR decreases S/G and also at an alternative site within the NS4A region. To determine the importance of specific residues within the consensus sequence for cleavage at the 2A/2B site, amino acid substitutions were introduced at each of the P4, P3, P2, P1, and P1' positions and the effects on proteolytic processing of a sig2A-5(356) polyprotein were examined using a vaccinia virus-T7 transient expression system. At the P1 and P1' positions, only the conservative substitutions P1:R-->K and P1':S-->G allowed efficient cleavage, suggesting that basic and small aliphatic amino acids are preferred at the P1 and P1' positions, respectively. At the P2 position, a preference for a basic amino acid was observed. In contrast, the P3 and P4 positions tolerated nonconservative substitutions and at P4 both enhancement and reduction in cleavage efficiency was observed. Evidence for cleavage at an alternative site within NS2A, defined by the sequence QK decreases T (NS2A residues 189-191) was obtained. Cleavage at this site, designated at NS2A alpha, is dependent upon an active NS2B-3 proteinase. To examine the effects of reduced cleavage efficiency at the 2A/2B and NS2A alpha cleavage sites on viral replication, mutations at each or both of these sites were incorporated into a full-length YF-17D cDNA template. RNA transcripts containing mutations which abolish cleavage were noninfectious whereas virus was recovered from several clones with mutations allowing partial cleavage at 2A/2B. However, some of these mutants exhibited a small plaque phenotype as well as reductions in RNA-specific infectivity and virus yield.
authors.affiliation: Department of Biochemistry and Molecular Biology, Australian National University, Canberra.
authors.forename: A
authors.initials: A
authors.lastname: Nestorowicz
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; D002460:Cell Line; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D015005:Yellow fever virus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D017361:Viral Nonstructural Proteins