title: Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities.
abstract: A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B-NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 degrees C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B-NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Avenue, St Louis, MO 63104, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Chambers
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Avenue, St Louis, MO 63104, USA.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Droll
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Avenue, St Louis, MO 63104, USA.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Avenue, St Louis, MO 63104, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Center for Macromolecular Crystallography, University of Alabama at Birmingham, 79-THT, MCLM-244, 1918 University Boulevard, Birmingham, AL 35294-0005, USA.
authors.forename: Vannakambadi K
authors.initials: VK
authors.lastname: Ganesh
authors.affiliation: Center for Macromolecular Crystallography, University of Alabama at Birmingham, 79-THT, MCLM-244, 1918 University Boulevard, Birmingham, AL 35294-0005, USA.
authors.forename: Krishna H M
authors.initials: KHM
authors.lastname: Murthy
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Avenue, St Louis, MO 63104, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Nickells
mesh_terms: D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010948:Viral Plaque Assay; D015005:Yellow fever virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases