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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. Sitedirected 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.Yellow fever virus (YF), the prototype member of the family Flaviviridae, contains a single molecule of positive-stranded RNA -11 kilobases long (1). The gene order is 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' where C, prM, and E denote the structural protein precursors and NS1 through NS5 represent the nonstructural proteins (NSs). A single long open reading frame encodes these proteins, which are produced by proteolytic cleavage (1, 2). It has been proposed that the structural protein precursors and the N terminus of NS4B are processed cotranslationally by host signalase in association with membranes of the endoplasmic reticulum (3,4 (8,9). The positions of three amino acid residues of YF NS3 (His-53, Asp-77, and Ser-138) are strictly conserved among flaviviruses and correspond spatially to the catalytic triad of the trypsin-like serine proteases.In this report we have obtained evidence for a protease activity encoded by the YF NS2B-NS3 region, mutagenized the histidine, aspartic acid, and serine residues in the proposed NS3 catalytic triad, and have studied the effects of mutations that abolish or diminish the in vitro cleavage activity on YF infectivity. MATERIALS AND METHODSCell Culture and Virus Infection. Growth of BHK-21 cell monolayers and their infection with the YF 17D strain were carried out as described (10).Construction of Transcription Vectors. DNA cloning was done using standard procedures (11). The transcription vector pET8C (12) contains a promoter for T7 RNA polymerase, followed by a unique Nco I site (CCATGG) with the ATG in an appropriate context for either prokaryotic or eukaryotic expression (12). Regions of YF cDNA were subcloned into pET8C using this Nco I site and a BamHI site preceding the T7 terminator (12). For construction of pET8C-NS2B3.1, YFM5.2 DNA (13) was subjected to polymerase chain reaction amplification using two synthetic oligonucleotide primers (14) that positione...

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The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the dibasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.