PMID:15831952

Citation	Chambers, TJ, Droll, DA, Tang, Y, Liang, Y, Ganesh, VK, Murthy, KH and Nickells, M (2005) Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities. J. Gen. Virol. 86:1403-13
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.
Links	PubMed Online version:10.1099/vir.0.80427-0
Keywords	Amino Acid Substitution; DNA Mutational Analysis; Humans; Models, Molecular; Mutagenesis, Site-Directed; Protein Structure, Tertiary; RNA Helicases/chemistry; RNA Helicases/genetics; RNA Helicases/metabolism; Serine Endopeptidases/chemistry; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism; Viral Nonstructural Proteins/chemistry; Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/metabolism; Viral Plaque Assay; Yellow fever virus/enzymology; Yellow fever virus/genetics; Yellow fever virus/growth & development
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
YEFV1:POLG		GO:0031079: picornain 3C activity	ECO:0000314:		F		Figure 5 showed that different mutations in this protein caused different effects in cleavage	complete CACAO 9334

References

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