Evidence that the N-Terminal Domain of Nonstructural Protein NS3 from Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 22; pp. 8898 - 8902
• Main Authors: Chambers, Thomas J., Weir, Ronald C., Grakoui, Arash, McCourt, David W., Bazan, J. Fernando, Fletterick, Robert J., Rice, Charles M.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.11.1990; National Acad Sciences
• Subjects: Amino acids; Biochemistry; Biological and medical sciences; Capsid - genetics; Capsid - metabolism; cloning vectors; Cloning, Molecular; DNA Mutational Analysis; Enzymes; Fundamental and applied biological sciences. Psychology; Genes, Viral; Genetic mutation; Microbiology; Morphology, structure, chemical composition, physicochemical properties; mutagenesis; nonstructural proteins; Polyproteins; Protein Biosynthesis; Protein Precursors - metabolism; Proteins; proteolysis; RNA; Serine Endopeptidases - genetics; transfection; translation; Viral Core Proteins - genetics; Viral Core Proteins - metabolism; Viral Nonstructural Proteins; Viral Proteins - metabolism; Viral Structural Proteins - genetics; Virology; Viruses; Yellow fever; Yellow fever virus; Yellow fever virus - enzymology; Yellow fever virus - genetics; Virus; Proteins; Molecular processing; Site directed mutagenesis; Flaviviridae; Flavivirus; Yellow fever virus
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.22.8898

Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavivirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cisacting site-specific proteolysis at the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis of the His-53, Asp-77, and Ser-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.