Hepatitis C virus escape from the interferon regulatory factor 3 pathway by a passive and active evasion strategy†
Version of Record online: 1 AUG 2007
Copyright © 2007 American Association for the Study of Liver Diseases
Volume 46, Issue 5, pages 1365–1374, November 2007
How to Cite
Binder, M., Kochs, G., Bartenschlager, R. and Lohmann, V. (2007), Hepatitis C virus escape from the interferon regulatory factor 3 pathway by a passive and active evasion strategy. Hepatology, 46: 1365–1374. doi: 10.1002/hep.21829
Potential conflict of interest: Nothing to report.
- Issue online: 29 OCT 2007
- Version of Record online: 1 AUG 2007
- Manuscript Accepted: 22 MAY 2007
- Manuscript Received: 27 FEB 2007
- National Genome Research Network, NGFN
- German Ministry for Research and Education and the “Kompetenznetz Hepatitis (Teilprojekt 13.2)”
Supplementary material for this article can be found on the H EPATOLOGY Web site ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).
|jws-hep.21829.fig1.pdf||162K||Supplementary Fig. 1Replication of a weakly adapted HCV replicon in cells with blocked IRF-3 activation. Replication efficiency of Con1/E1202G+T1280I (dark bars) in Huh7-Lunet and Huh-7 WU cells stably transduced with empty vector, RIG-I dominant negative (dn) or HCV NS3/4A protease. Background reporter activity of a non-replicating control (Con1/lucΔGDD, light bars) is given for reference. Values are relative light units (RLU) at 48 hours normalized to the input measured at 4 hours and are displayed on a logarithmic scale. Error bars are standard deviations of duplicate measurements in duplicate wells.|
|jws-hep.21829.fig2.pdf||278K||Supplementary Fig. 2HCV replication in Huh7.5 cells with reconstituted RIG-I signaling. (A) Replication of weakly adapted Con1/E1202G+T1280I replicon (dark bars) in Huh7.5 cells stably transduced with empty vector, myc-RIG-I wildtype (wt) or myc-RIG-I constitutively active (ca). Background luciferase activity of a non-replicating control (Con1/lucΔGDD, light bars) is given for reference. Values are relative light units (RLU) at 48 hours normalized to the input measured at 4 hours and are displayed on a logarithmic scale. Error bars are standard deviations of duplicate measurements in duplicate wells. (B) Immunofluorescence analysis of Huh7.5 cells stably transduced with empty vector, myc-RIG-I wildtype (wt) or myc-RIG-I constitutively active (ca). RIG-I was detected using α-myc antibody (red channel) and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue signal). Cells were uninfected (-) or infected (+) with Jc1/GFP, expressing a GFP-tagged NS5A (green channel). Yellow arrowheads point to cells positive for both, myc and NS5A.|
|jws-hep.21829.fig3.pdf||226K||Supplementary Fig.3 IRF-3 activation upon transfection of replicating HCV RNA. (A) ISG56 promoter reporter activity in Huh7.5 RIG-I wt cells upon cotransfection of poly(C) (blue line) or poly(I:C) (red line) together with Con1/Rluc RNA. Cells were harvested 4, 8, 12, 24 and 48 hours after transfection and firefly luciferase activity was measured. RLU are normalized to β-galactosidase reporter activity and are expressed as fold-increase over poly(C) (without HCV) transfection. RLU are mean values of duplicate measurements of one representative experiment. Error bars are standard deviations. (B) Con1/Rluc replication in the same experiment as in panel (A). HCV replication was determined by measuring Renilla luciferase activity. RLU were normalized to β-galactosidase activity and are expressed as mean values of duplicate measurements of one representative experiment. Standard deviations of the measurements are included in the graph but too small to be seen. Data are displayed on a logarithmic scale.|
|jws-hep.21829.fig4.pdf||259K||Supplementary Fig. 4Schematic representation of RIG-I signaling in absence or presence of HCV. (A) Simplified scheme of IRF-3 activation in the absence of HCV. Double-stranded or 5?-triphosphorylated inducer RNA activates RIG-I which associates with the adaptor protein Cardif. This leads to the recruitment of cellular kinases including IKK&#949; and TBK1, which in turn phosphorylate the transcription factor IRF-3. Phosphorylated IRF-3 dimerizes and translocates to the nucleus where, in conjunction with additional transcription factors, it promotes the expression of antiviral effector genes like interferon β (IFN-β) and a plethora of different interferon stimulated genes (ISGs). (B) Schematic representation of our proposed model on the interplay of HCV with the RIG-I pathway. Upon entry, single stranded HCV genomes fail to efficiently induce RIG-I signaling. Second, the incoming RNA is heavily translated, giving rise to an excess of viral proteins, including NS3/4A protease which in turn depletes the cell of functional Cardif. Subsequently, RNA replication commences, liberating offspring HCV genomes and putatively also double-stranded replication intermediates. Activation of RIG-I by any product of viral replication, however, will not lead to IRF-3 phosphorylation due to the lack of functional Cardif which is essential for transmitting the signal.|
|jws-hep.21829.mat1.pdf||57K||Supporting Information file jws-hep.21829.mat1.pdf|
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