Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone.
Hepatitis C virus (HCV) nonstructural protein 3 (NS3), with its protease, helicase, and NTPase enzymatic activities, plays a crucial role in viral replication, and therefore represents an ideal target for the development of anti-viral agents. We have developed a recombinant human antibody (Fab) that reacts with the helicase domain of HCV NS3. The affinity-purified Fab antibody completely inhibited the helicase activity of HCV NS3 at equimolar concentration. To evaluate the effect of the Fab on HCV replication, the clone encoding the Fab gene was put into an expression vector, which converts Fab into a complete IgG1 antibody. Using a DNA-based transfection model, we demonstrated that intracellular expression of this antibody resulted in significant reduction of HCV-negative strand RNA synthesis. Intracellular expression of this antibody into either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. These results support the use of recombinant antibody fragments to inhibit NS3 enzyme as a novel, feasible, and effective approach for inhibiting HCV replication.
Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Ave., New Orleans LA 70112, USA.
Ramesh
R
Prabhu
Nutan
N
Khalap
Roberto
R
Burioni
Massimo
M
Clementi
Robert F
RF
Garry
Srikanta
S
Dash
D000818:Animals; D000915:Antibody Affinity; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D002999:Clone Cells; D016174:Hepacivirus / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D007140:Immunoglobulin Fab Fragments; D012313:RNA; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases