Inhibition of HCV replication by humanized single domain transbodies to NS4B.
NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti viral immunity. Thus, NS4B is an attractive target of a novel anti HCV agent. In this study, humanized nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN) for making them cell penetrable (become transbodies) Human hepatic (Huh7) cells transfected with HCV JFH1 RNA that were treated with transbodies from four Escherichia coli clones (PEN VHH7, PEN VHH9, PEN VH33, and PEN VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNb and IL 28b. The transbodies have high potential for testing further as a novel anti HCV agent, either alone, adjunct of existing anti HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.
Biochemical and biophysical research communications