Complex Formation between the Hepatitis C Virus Serine Protease and A Synthetic NS4A Cofactor Peptide

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K i values as low as 3 M. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain

Section: Table Imentioning

confidence: 42%

Inhibition of Hepatitis C Virus NS2/3 Processing by NS4A Peptides

Darke

Jacobs

Waxman

et al. 1999

“…To this purpose, the effects of complex formation with the NS4A cofactor peptide and with a substrate peptide have been studied. Both substrate and co-factor binding have been shown to be glycerol-dependent (11). This precluded observation by NMR due to the excessive line-broadening caused by the high viscosity of glycerol-containing buffer solutions.…”

Section: Resultsmentioning

confidence: 99%

The Metal Binding Site of the Hepatitis C Virus NS3 Protease

Urbani¹,

Bazzo²,

Nardi³

et al. 1998

Self Cite

The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines (Cys 97 has shown the importance of this residue in the metal incorporation pathway and for achieving an active fold. The metal coordination of the protease was also investigated by circular dichroism and electronic absorption spectroscopies using a Co(II)-substituted enzyme. We show evidence for rearrangements of the metal coordination geometry induced by complex formation with an NS4A peptide cofactor. No such changes were observed upon binding to a substrate peptide. Also, CN ؊ and N 3 ؊ induced Co(II) ligand field perturbations, which went along with an 1.5-fold enhancement of protease activity.The hepatitis C Virus (HCV) 1 has been identified as the major etiologic agent of parenterally transmitted non-A non-B hepatitis (1, 2). HCV is an enveloped virus with a positivestranded RNA genome of 9.4 kb, which is translated into a precursor polyprotein of about 3010 amino acids (3). Both cellular and virally encoded proteases are involved in the maturational proteolytic processing of this precursor. Whereas the structural viral proteins arise from signal peptidase-catalyzed cleavages (4), two different proteolytic activities encoded by the HCV NS2 and NS3 proteins are responsible for the processing of the nonstructural region of the polyprotein. The NS2-NS3 precursor is cleaved intramolecularly by an autoprotease, the activity of which was shown to be zinc-dependent (5). The N-terminal part of the NS3 protein, furthermore, contains a 20-kDa serine protease domain that accomplishes all cleavage events downstream of NS3, including the generation of the mature viral polymerase (6). In order to perform its physiological task, the NS3 serine protease has to bind to the viral protein NS4A (7,8). This binding event leads to an enhancement of the protease activity and to a stabilization of NS3. In vitro, activation of NS3 can be achieved by addition of peptides harboring residues 21-34 of NS4A (9 -12).Based on a homology model, we were able to predict the presence, in the NS3 protease domain, of a tetradentate metal binding site formed by three cysteines (Cys 97 , Cys 99 , and Cys 145 ) and one histidine residue (His 149 ) (13). Biochemical characterization has confirmed this prediction and demonstrated the presence of a zinc ion in a tetrahedral environment (13,14). This zinc ion was shown to be essential for the structural integrity of the protein; its removal leads to unfolding and aggregation of the enzyme. Mutagenesis experiments have shown that mutations affecting any of the three cysteine residues resulted in an impaired NS3 protease activity as judged from in vitro translation experiments (14, 15). On the other hand, mutagenesis...

“…2). This additional band must result from emission of the dansyl moiety upon energy transfer from the NS3 tryptophan residue(s), which emit at 330 nm (15). Unbound compound P showed no significant fluorescence at 510 nm if excited at 280 nm in the absence of NS3 (not shown).…”

Section: Resultsmentioning

confidence: 94%

“…In vitro, activation can be obtained by the addition of synthetic peptides encompassing residues 21-34 of the NS4A cofactor to the purified enzyme (11)(12)(13)(14)(15). The x-ray crystal structures of the uncomplexed protease domain and of the binary serine proteasecofactor peptide complex have been determined (16 -18).…”

Section: The Ns3 Protein Of the Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Probing the Active Site of the Hepatitis C Virus Serine Protease by Fluorescence Resonance Energy Transfer

Fattori¹,

Urbani²,

Brunetti³

et al. 2000

Self Cite

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding. The NS3 protein of the hepatitis C virus (HCV)1 is a multifunctional polypeptide. Its C-terminal two-thirds encompasses an RNA helicase, thought to be involved in the separation of RNA double strand structures that transiently form during the replication of the viral genome (1, 2). The N-terminal third of the protein, instead, harbors a serine protease domain responsible for proteolytic maturation of most of the nonstructural region of the viral polyprotein (reviewed in Ref.3). Its activity is thought to be absolutely required for the assembly of the viral replication machinery. Both enzymatic functions are presently the focus of intensive research, since their inhibition is considered as a possible strategy for the development of antiviral pharmaceuticals (4 -6).The NS3 protein shows only very weak proteolytic activity on its own and complex formation with the viral protein NS4A is required to activate its catalytic functions (7-10). In vitro, activation can be obtained by the addition of synthetic peptides encompassing residues 21-34 of the NS4A cofactor to the purified enzyme (11-15). The x-ray crystal structures of the uncomplexed protease domain and of the binary serine proteasecofactor peptide complex have been determined (16 -18). Based on these structures, it was proposed that the cofactor activates the enzyme by promoting a correct positioning of the residues that form its catalytic triad (19).Both kinetic and structural data have shed light on the mechanisms by which the enzyme recognizes its substrates (13, 20 -22). The minimum length for peptide substrates was shown to be a decamer spanning from P6 to P4Ј (13). This requirement for rather large substrate molecules stems from the peculiar architecture of the substrate binding site that lacks all major loops that form the S2 or S3 binding pockets in other enzymes of this family. Sp...