Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase.

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS: We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS: The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).

Human interleukin 4 receptor complex: neutralization effect of two monoclonal antibodies.

The interaction of human interleukin 4 with the extracellular domain of its receptor alpha-subunit (shuIL-4R alpha) was characterized in studies utilizing chemical cross-linking, size exclusion chromatography, and Western blot analysis. A 1:1 stoichiometric complex could be demonstrated over a wide range (0.04-2.7) of ligand-receptor concentration ratios. It could also be cross-linked with bifunctional reagents containing a minimum chain length of eight methylene residues or the equivalent (11.4 angstroms). Using surface plasmon resonance, (SPR) technology, we established the high-affinity of human interleukin 4 (huIL-4) to shuIL-4R alpha which was immobilized on a BIAcore sensor chip (K(d) = 46 pM). The mechanisms of action of neutralizing monoclonal antibodies (Mab) 25D2 and 35F2 [Abrams et al. (1991) U.S. Patent 5,041,381; Ramanathan et al. (1990) in Advances in Gene Technology: The Molecular Biology of Immune Diseases and the Immune Response (Streilein, J. W., et al., Eds.) p 163, IRL Press, Oxford; DeKruyff et al. (1989) J. Exp. Med. 170, 1477-1493] were subsequently evaluated on the basis of their interaction with huIL-4 in the presence of shuIL-4R alpha. SPR studies showed that Mab 25D2 binds to huIL-4 and reduces its affinity for shuIL-4R alpha by 54-fold. Formation of a ternary complex between Mab 25D2 and the huIL-4/shuIL-4R alpha complex was demonstrated in size exclusion chromatography experiments. In contrast, Mab 35F2 which also binds huIL-4 failed to form a stable ternary complex with huIL-4 and shuIL-4 alpha during size exclusion chromatography. SPR studies supported this finding and showed that the interactions of Mab 35F2 and shuIL-4R alpha to huIL-4 are mutually exclusive. These data are consistent with results of previous epitope mapping studies showing that Mabs 25D2 and 35F2 bind to huIL-4 at two different sites [Ramanathan et al. (1993) Biochemistry 32, 3549-3556]. Together, the results suggest that Mab 25D2 binds to a domain in huIL-4 including helix D and exerts its inhibitory effect through a dual action. It decreases the affinity of huIL-4 for huIL-4R alpha and potentially blocks interaction with a secondary receptor subunit such as the IL-2R gamma [Reusch et al. (1994) Eur. J. Biochem. 222, 491-499]. Mab 35F2 operates through a direct and simpler mechanism, binding to helix C and inhibiting huIL-4 activity by sterically excluding all interaction with huIL-4R alpha.

Development of a receptor peptide antagonist to human gamma-interferon and characterization of its ligand-bound conformation using transferred nuclear Overhauser effect spectroscopy.

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

A role for the carboxyl terminus of human granulocyte-macrophage colony-stimulating factor in the binding of ligand to the alpha-subunit of the high affinity receptor.

A synthetic segment (110-127) of the carboxyl terminus of recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was used to generate a rabbit polyclonal antibody (345-6), which recognized both peptide and full-length Escherichia coli-derived rh-GM-CSF in a direct enzyme-linked immunosorbent assay. Antibody 345-6 was shown to antagonize the binding of 125I-labeled rh-GM-CSF to its receptor on the KG-1 cell line and to inhibit human GM-CSF-dependent proliferation of the AML-193 cell line. The purified IgG fraction of neutralizing antibody 345-6 was used as immunogen to obtain sheep anti-serum 1418. Antibody 1418 recognized antibody 345-6 on direct enzyme-linked immunosorbent assay but did not recognize rh-GM-CSF or the peptide 110-127 to which antibody 345-6 was raised. Antiserum 1418, as well as a purified IgG fraction of this serum, inhibited both rh-GM-CSF-stimulated cell proliferation and 125I-labeled rh-GM-CSF receptor binding but not 125I-labeled recombinant human interleukin-4 receptor binding. The anti-idiotypic antibody response derived from the anti-(110-127) antibody strongly suggests that the carboxyl-terminal region of rh-GM-CSF may be directly involved in the receptor-ligand interaction of this protein. The high affinity receptor consists of two different components (GM-R alpha beta) a cytokine-specific alpha-subunit and a beta-subunit that is shared by human GM-CSF, interleukin-3, and interleukin-5. In an effort to localize the epitope of antibody 1418 to either GMR alpha or GMR beta, several cell lines containing high, low, or both high and low affinity receptors were examined. Each was specifically and completely inhibited by antibody 1418. Interleukin-3-dependent cell proliferation of the AML-193 cell line was found to be unaffected by the antibody 1418. Thus, the carboxyl-terminal region of rh-GM-CSF is likely to be involved in the interaction of the ligand with the alpha-subunit of the high affinity receptor.

Synthetic mimics of juxtaposed amino- and carboxyl-terminal peptide domains of human gamma interferon block ligand binding to human gamma interferon receptor.

The epitopes of two neutralizing antibodies (47N3-6 and 47N30A35) raised against rhuIFN-gamma each mapped both to amino-terminal regions (22-29 and 12-19, respectively) and to a carboxyl-terminal region 131-139, suggesting the juxtaposition of these two domains in the native protein. Three novel peptides were designed to mimic a conformation of rhuIFN-gamma that places the two regions in close proximity (discontinuous peptides 1 (15-21-GGG-132-138), 2 (15-29...111-118...130-138), and 3 (15-21-CGPGC-130-138)), by bridging the amino- and carboxyl-terminal regions of gamma interferon. Each discontinuous peptide inhibits biological or receptor binding activities with an IC50 of 15-50 microM and produces a neutralizing antibody when used as an immunogen. Neutralizing rabbit polyclonal antibody (P616) raised against discontinuous peptide 1 was used as immunogen to generate an anti-idiotypic response. This anti-idiotypic antibody inhibits receptor binding and recognizes soluble gamma interferon receptor on direct enzyme-linked immunosorbent assay. The anti-idiotypic response suggests that juxtaposed regions at the amino and carboxyl termini serve as the receptor-ligand binding site of human gamma interferon.

Identification of functionally distinct domains of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.