Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation.

Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFκB promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

[1]  S. Lemon,et al.  Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies. , 2010, Antiviral research.

[2]  S. Chen,et al.  Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[3]  D. Lavanchy,et al.  Evolving epidemiology of hepatitis C virus. , 2011, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[4]  M. Lai,et al.  Hepatitis C virus core protein: possible roles in viral pathogenesis. , 2000, Current topics in microbiology and immunology.

[5]  Mitsuru Sato,et al.  Single domain intrabodies against WASP inhibit TCR-induced immune responses in transgenic mice T cells , 2013, Scientific Reports.

[6]  K. Ishii,et al.  Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription , 2010, Journal of Virology.

[7]  J. Dubuisson,et al.  CD81 Expression Is Important for the Permissiveness of Huh7 Cell Clones for Heterogeneous Hepatitis C Virus Infection , 2007, Journal of Virology.

[8]  K. Shimotohno,et al.  Hepatitis C Virus Core Protein Inhibits Fas- and Tumor Necrosis Factor Alpha-Mediated Apoptosis via NF-κB Activation , 1999, Journal of Virology.

[9]  Haruo Watanabe,et al.  Evaluation of Hepatitis C Virus Core Antigen Assays in Detecting Recombinant Viral Antigens of Various Genotypes , 2009, Journal of Clinical Microbiology.

[10]  Yan-Hwa Wu Lee,et al.  Hepatitis C Virus Core Protein Enhances NF-κB Signal Pathway Triggering by Lymphotoxin-β Receptor Ligand and Tumor Necrosis Factor Alpha , 1999, Journal of Virology.

[11]  Austin Hughes,et al.  A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks , 1995, Nature.

[12]  K. Sherman,et al.  Prevalence of mutations in hepatitis C virus core protein associated with alteration of NF-kappaB activation. , 2006, Virus research.

[13]  K. Shimotohno,et al.  Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation. , 1999, Journal of virology.

[14]  S. Muyldermans,et al.  Naturally occurring antibodies devoid of light chains , 1993, Nature.

[15]  Y. Kanegae,et al.  Possible Mechanism of Adenovirus Generation from a Cloned Viral Genome Tagged with Nucleotides at Its Ends , 2006, Microbiology and immunology.

[16]  Roger L. Williams,et al.  Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS , 2007, The EMBO journal.

[17]  H. de Haard,et al.  Properties, production, and applications of camelid single-domain antibody fragments , 2007, Applied Microbiology and Biotechnology.

[18]  K. Ishii,et al.  Trans-complemented hepatitis C virus particles as a versatile tool for study of virus assembly and infection. , 2012, Virology.

[19]  Y. Lee,et al.  Hepatitis C virus core protein enhances NF-kappaB signal pathway triggering by lymphotoxin-beta receptor ligand and tumor necrosis factor alpha. , 1999, Journal of virology.

[20]  B. Lindenbach,et al.  Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly , 2011, PLoS pathogens.

[21]  T. Rabbitts,et al.  Intracellular antibody capture: A molecular biology approach to inhibitors of protein-protein interactions. , 2014, Biochimica et biophysica acta.

[22]  Jun Kato,et al.  Hepatitis C Virus Core Protein Activates Nuclear Factor κB-dependent Signaling through Tumor Necrosis Factor Receptor-associated Factor* , 2001, The Journal of Biological Chemistry.

[23]  C. Barbas,et al.  Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells. , 2005, Blood.

[24]  R. Ray,et al.  Hepatitis C virus core protein: intriguing properties and functional relevance. , 2001, FEMS microbiology letters.

[25]  N. Hayashi,et al.  Intracellular single‐chain antibody against hepatitis B virus core protein inhibits the replication of hepatitis B virus in cultured cells , 1999, Hepatology.

[26]  K. Ishii,et al.  Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles , 2008, Journal of Virology.