Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein

Abstract

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domainspecific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.The hepatitis C virus (HCV) nonstructural 3 protein (NS3) performs vital enzymatic functions in the HCV life cycle. The N-terminal one-third serine-like protease domain of the NS3 protein, together with the cofactor NS4A, is required for cleavage of the junction between NS2 and NS3 and the downstream NS3/NS4A, NS4A/NS4B, NS4B/NSA5, and NS5A/NS5B junctions (1, 4). The C-terminal two-thirds of the NS3 protein has NTPase and helicase functions (6, 16). Importantly, the NS3 protein is also one of most conserved proteins of HCV. Collectively, these properties suggest that the NS3 protein may be a suitable target for antiviral therapy.The variable regions of the heavy and light chains (VH and VL, respectively) of an antibody are essential for the recognition of antigens. Single-chain variable-region fragments (ScFvs) of antibodies, containing VH and VL DNA assembled by a flexible linker sequence, allow for intracellular expression of antibody fragments. Several reports have demonstrated that intracellularly expressed ScFvs may reduce the susceptibility of transfected cells to viral infections or disturb viral enzymes within the life cycles of human immunodeficiency virus type (9, 20), tick-borne encephalitis virus (5), and murine coronavirus (8). The production of ScFvs to the HCV core and envelope proteins was recently described (2). This approach therefore offers an alternative antiviral therapy by blocking vital steps in the viral life cycle inside infected cells. We have developed a murine anti-NS3 monoclonal antibody (MAb) which recognizes the NS3 ATPase/helicase domain. From the NS3-specific MAb we designed and produced a functional NS3-specific ScFv by use of procaryotic and eucaryotic expression systems.

Molecular Basis for the Interaction of the Hepatitis B Virus Core Antigen with the Surface Immunoglobulin Receptor on Naive B Cells

“…The VH and VL domains of MAbs were amplified from cDNA by PCR using the primer sequences designed for PCR-based cloning of VH and VL regions (14a) and Ig-prime kits (Novagen, Madison, Wis.). The amplified cDNA fragments were directly ligated into the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.) as previously described (28). The DNA sequence was determined by an automated sequencer (ALF Express; Pharmacia, Uppsala, Sweden) as previously described (28).…”

“…The amplified cDNA fragments were directly ligated into the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.) as previously described (28). The DNA sequence was determined by an automated sequencer (ALF Express; Pharmacia, Uppsala, Sweden) as previously described (28).…”

“…Total cellular mRNA was extracted by using magnetic beads coated with oligo-dT25 (Dynal AS, Oslo, Norway) as previously described (28). The VH and VL domains of MAbs were amplified from cDNA by PCR using the primer sequences designed for PCR-based cloning of VH and VL regions (14a) and Ig-prime kits (Novagen, Madison, Wis.).…”

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

“…A human monoclonal antibody CM3.B6 recognized a major epitope located at the minimal residues 1378–1443 within c33c [4]. A murine mAb ZX10 recognized a discontinuous epitope of NS3 helicase domain, encompassing residues 1371 to 1382 [5]. However, monoclonal antibodies to NS3 helicase have been neither generated nor explored extensively for epitope identification and impact of monoclonal antibodies on its enzymatic function in HCV infection.…”

The Hepatitis C Virus NS3 Protein: A Model RNA Helicase and Potential Drug Target

“…Tessman et al have used this technique to isolate a series of high affinity ScFv's that specifically interact with HCV helicase (Tessmann et al, 2002). ScFv's that bind HCV helicase have also been constructed by splicing together the variable domains of monoclonal antibodies (Zhang et al, 2000;Sullivan et al, 2002), and after expression and purification, several of these recombinant proteins inhibit HCV helicase-catalyzed DNA unwinding (Sullivan et al, 2002;Artsaenko et al, 2003). One particular ScFv consists of the variable regions of the human monoclonal antibody CM3.B6, which recognizes an epitope that spans conserved SF2 helicase motifs IV and V (Mondelli et al, 1994).…”