Distinct antiviral signaling pathways in primary human hepatocytes and their differential disruption by HCV NS3 protease
Received 30 June 2009; received in revised form 28 August 2009; accepted 1 September 2009. published online 27 November 2009.
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.
1Laboratoire d’immunologie virale, Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, 264 René Levesque-Est, PEA 312, Montréal, Québec, Canada H2X 1P1
2Laboratoire d’hépatologie cellulaire, Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada H2X 1P1
3Laboratoire de signalisation et infections virales, Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada H2X 1P1
4Département de Sciences Biomédicales, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7
5Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7
6Département de Médecine, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7
7Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7
8Institut de Recherche en Immunologie et en Cancérologie, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7
Corresponding author. Address: Laboratoire d’immunologie virale, Centre de Recherche du CHUM (CRCHUM), Hôpital Saint-Luc, 264 René Levesque-Est, PEA 312, Montréal, Québec, Canada H2X 1P1. Tel.: +1 514 343 7127; fax: +1 514 343 2165.
ABSTRACT