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.

Abstract

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...

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

“…By these methods, it has been established that the NS3 region of the polyprotein contains a serine proteinase domain which is responsible for cleavage of the downstream polyprotein in at least four places (Bartenschlager et al, 1993(Bartenschlager et al, , 1994Eckart et al, 1993;Grakoui et al, 1993;Hijikata et al, 1993;Tomei et al, 1993 ;Manabe et al, 1994). By analogy with the yellow fever virus (Chambers et al, 1990) this proteinase is probably essential for the production of infectious virus, and hence represents a possible target for chemotherapeutic intervention.…”

Characterizationof the NTPase Activity of Japanese Encephalitis Virus NS3 Protein

“…The JE virus genome consists of a positive-sense, single-stranded RNA molecule of approximately 1I kb and encodes a single polycistronic message which is translated into a polyprotein precursor. Processing by cellular and virus proteases (Bazan & Fletterick, 1989;Ruiz-Linares et aI., 1989;Chambers et al, 1990;Preugschat et al, 1991;Wengler et al, 1991) produces three structural proteins-the capsid (C), the precursor to the membrane (prM) and the envelope (E) proteins-and seven nonstructural proteins-NS1 through NS5 (Sumiyoshi eta]., 1987; Nitayaphan et al, 1990;Hashimoto eta]., 1990).…”

Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis

“…The serine protease domain is located at the amino terminus of the protein (2,10). The proteases of DEN, YF, West Nile, Murray Valley, and TBE viruses have all been shown to have activity (5,20,23,26,33). A catalytic triad of amino acids, His-AspSer, is essential for protease activity in vitro and for viral replication (5,20,30,33).…”

“…The proteases of DEN, YF, West Nile, Murray Valley, and TBE viruses have all been shown to have activity (5,20,23,26,33). A catalytic triad of amino acids, His-AspSer, is essential for protease activity in vitro and for viral replication (5,20,30,33). The proteolytic function of the NS3 protein is dependent on the presence of NS2B as a cofactor and is associated with the membrane fraction of infected cells (6).…”