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.
Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus 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 cis-acting 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.
Chambers
T J
TJ
Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110-1093.
Weir
R C
RC
Grakoui
A
A
McCourt
D W
DW
Bazan
J F
JF
Fletterick
R J
RJ
Rice
C M
CM
D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005814:Genes, Viral; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D015005:Yellow fever virus / Q000201:enzymology* / Q000235:genetics
D011498:Protein Precursors; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D012697:Serine Endopeptidases