Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies.
Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.
Gal-Tanamy
Meital
M
Molecular Hepatology Research Laboratory, Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Petah Tikva, Israel.
Zemel
Romy
R
Bachmatov
Larissa
L
Jangra
Rohit K
RK
Shapira
Assaf
A
Villanueva
Rodrigo A
RA
Yi
Minkyung
M
Lemon
Stanley M
SM
Benhar
Itai
I
Tur-Kaspa
Ran
R
D000914:Antibodies, Viral / Q000276:immunology; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D004359:Drug Therapy, Combination; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000502:physiology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology / Q000627:therapeutic use; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020815:Polyproteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase; D012093:Replicon / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000627:therapeutic use*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000914:Antibodies, Viral; D000998:Antiviral Agents; D016898:Interferon-alpha; D020815:Polyproteins; D011480:Protease Inhibitors; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D057127:Single-Chain Antibodies; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases