Distinct antiviral signaling pathways in primary human hepatocytes and their differential disruption by HCV NS3 protease

Background & Aims

Molecular sensors recognize viral nucleic acids and initiate events that subsequently enable cells to control and clear infection. Hepatitis C Virus (HCV) can interfere with the innate host response and the NS3/4A protease was reported to specifically block antiviral signaling pathways, a finding that had yet to be studied in human primary hepatocytes.

Methods

Freshly isolated human primary hepatocytes, transduced with a lentiviral vector expressing HCV NS3/4A were stimulated with extracellular and intracellular double-stranded RNA (dsRNA) and the innate immune antiviral genes were quantified by quantitative PCR and microarrays analysis.

Results

We demonstrate that sensing receptors of human hepatocytes in primary cultures are stimulated following recognition of either mode of dsRNA delivery, inducing transcriptional up-regulation (over 100-fold) of multiple immune genes, either selectively or independently of recognition pathways. We also report that the intracellular dsRNA-activated innate response is severely compromised upon ectopic expression of the HCV NS3/4A protease gene in normal human primary hepatocytes, and completely restored by treatment with the NS3/4A protease specific inhibitor BILN2061.

Conclusions

The present study indicates that NS3/4A has a wider protease-dependent effect on the intracellular Pathogen Recognition Receptor (PRR)-mediated immune response than on its extracellular counterpart, which underlies the major role of cytosolic dsRNA receptors in HCV recognition by primary human hepatocytes.

Abbreviations: HCV, hepatitis C virus, NS3/4A, non structural proteins 3 and 4A, PRR, pathogene recognition receptor, MOI, multiplicity of infection, edsRNA, extracellular double-stranded ribonucleic acid, idsRNA, intracellular double-stranded ribonucleic acid, TLR3, Toll-like receptor 3, RIG-I, retinoic acid inducible gene-I, IFN, interferon, ISG, interferon stimulated gene, ISRE, interferon-stimulated response element

Keywords: Hepatitis C virus, Primary hepatocytes, NS3/4A, Interferon stimulated genes, Viral interference