Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.Hepatitis C virus (HCV), a recently identified agent of parenterally transmitted non-A non-B hepatitis, causes the vast majority of transfusion-associated cases of hepatitis and a significant proportion of community-acquired hepatitis (for review, see refs. 1 and 2). HCV infection results in varied clinical outcomes, and chronic infections are common and have been associated with an increased incidence of hepatocellular carcinoma. Although a interferon has been partially successful in inhibiting HCV replication, reliable therapeutic agents for controlling or eradicating HCV infection are currently lacking.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.

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...

The hepatitis C virus (HCV) H strain polyprotein is cleaved to produce at least nine distinct products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. In this report, a series of C-terminal truncations and fusion with a human c-myc epitope tag allowed identification of a tenth HCV-encoded cleavage product, p7, which is located between the E2 and NS2 proteins. As determined by N-terminal sequence analysis, p7 begins with position 747 of the HCV H strain polyprotein. p7 is preceded by a hydrophobic sequence at the C terminus of E2 which may direct its translocation into the endoplasmic reticulum, allowing cleavage at the E2/p7 site by host signal peptidase. This hypothesis is supported by the observation that cleavage at the E2/p7 and p7/NS2 sites in cell-free translation studies was dependent upon the addition of microsomal membranes. However, unlike typical cotranslational signal peptidase cleavages, pulse-chase experiments indicate that cleavage at the E2/p7 site is incomplete, leading to the production of two E2-specific species, E2 and E2-p7. Possible roles of p7 and E2-p7 in the HCV life cycle are discussed.

Enterokinase is a protease of the intestinal brush border that specifically cleaves the acidic propeptide from trypsinogen to yield active trypsin. This ITPK-IVGG (human) or VSPK-IVGG (bovine), suggesting that single-chain enterokinase is activated by an unidentified trypsin-like protease that cleaves the indicated Lys-fle bond. Therefore, enterokinase may not be the "first" enzyme of the intestnal digestive hydrolase cascade. The specificity of enterokinase for the DDDDK-I sequence of trpsinogen may be explained by complementary basic-amino acid residues clustered in potential S2-S5 subsites.All animals need to digest exogenous macromolecules without destroying similar endogenous constituents. The regulation of digestive enzymes is, therefore, a fundamental requirement (1). Vertebrates have solved this problem, in part, by using a two-step enzymatic cascade to convert pancreatic zymogens to active enzymes in the lumen of the gut. The basic features ofthis cascade were described in 1899 by N. P. Schepovalnikov, working in the laboratory of I. P. Pavlov (2). Extracts of the proximal small intestine were shown to strikingly activate the latent hydrolytic enzymes in pancreatic fluid. Pavlov considered this intestinal factor to be an enzyme that activated other enzymes, or a "ferment of ferments," and named it "enterokinase." The importance of this protease cascade is emphasized by the life-threatening intestinal malabsorption that accompanies congenital deficiency of enterokinase (3, 4).Enterokinase activates bovine trypsinogen by cleaving after the sequence VDDDDK, releasing an amino-terminal activation peptide (5,6). The acidic DDDDK sequence of the trypsinogen-activation peptide is conserved among vertebrates (7), except for the similar sequences of trypsinogens from lungfish (IEEDK and LEDDK) and African clawed frog (FDDDK). Enterokinase prefers substrates with the sequence DDDDK, whereas the presence of aspartate residues markedly inhibits the ability of trypsin to cleave such substrates (8). For example, toward bovine trypsinogen the catalytic efficiency of enterokinase is 12,000-fold (porcine) (9) or 34,000-fold (bovine) (10) greater than that of bovine trypsin. This reciprocal specificity protects trypsinogen against autoactivation by trypsin and promotes activation by enterokinase in the gut.Enterokinase has been purified from porcine (11), bovine (10, 12, 13), human (14), and ostrich intestine (15). With the possible exception of human enterokinase, which was suggested to be a heterotrimer (14), enterokinase appears to be a disulfide-linked heterodimer with a heavy chain of 82-140 kDa and a light chain of 35-62 kDa. Mammalian enterokinases contain 30-50%o carbohydrate, which may contribute to the apparent differences in polypeptide masses. The heavy chain is postulated to mediate association with the intestinal brush border membrane (16), although no direct evidence for this function has been reported. The light chain contains the catalytic center. Based on susceptibility to inhibition by chem...