Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity

Abstract

NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this study, recombinant C-terminal NS3 protein of HCV genotype 3a with endowed helicase activity was produced and used as antigen by selecting VH/V(H)H display phage clones from an established humanized-camel single domain antibody library that bound specifically to HCV helicase. The VH/V(H)H derived from phage transfected Escherichia coli clones were linked molecularly to a cell penetrating peptide, i.e., penetratin (PEN). The cell penetrable VH/V(H)H (transbodies) could reduce the amounts of the HCV RNA released into the cell culture fluid and inside Huh7 cells infected with pJFH1 replicon with a greater effect on the former compared to the latter. Regions and residues of the helicase bound by the transbodies were determined by phage mimotope searching and multiple alignments as well as homology modeling and molecular docking. The epitope of one transbody (PEN-V(H)H9) encompassed residues 588RLKPTLHGPTPLLYRLGA605 of the domain 3 necessary for helicase activity while another transbody (PEN-VH59) interacted with the areas covering the phenylalanine loop and arginine clamp of the domain 2 which are important for the proper folding of the enzyme as well as nucleic acid substrate binding. Although the molecular mechanisms of the prototypic transbodies on NS3 helicase need further investigation, these transbodies have high potential as novel, safe and mutation tolerable anti-HCV agents.

Keywords: C-terminal at two-thirds of the non-structural NS3 protein of hepatitis C virus; Cell penetrable antibody (transbody); Epitope; FRET; HCV; Hepatitis C virus (HCV) helicase; Humanized-camel VH/V(H)H; NS3-C; OD; PCR; RNA; RT-PCR; V(H)H; VH; Virus neutralization assay; cDNA; complementary deoxyribonucleic acid; fluorescent resonance energy transfer; hepatitis C virus; optical density; polymerase chain reaction; reverse transcription polymerase chain reaction; ribonucleic acid; variable heavy chain domain; variable heavy chain domain of the heavy chain antibody.