Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells - PubMed (original) (raw)
Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells
Erika A Eksioglu et al. PLoS One. 2011.
Abstract
Background and aim: The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.
Methods: The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.
Results: HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.
Conclusion: These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. IFN response is dependent on viral replication.
A) IFNβ mRNA expression was measured daily from the total RNA of LH86 cells treated with an MOI of 0.1 of HCV/JFH-1. The expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. The “No virus” control indicates cells that were cultured with uninfected Huh7.5 supernatant, “HCV” is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1), “heated HCV” is the same supernatant as “HCV” but heated for 15 minutes at 72°C and “UV-treated HCV” was exposed to UV light for 15 minutes. B) IFNβ mRNA expression of different dilutions of virus (1∶1 MOI = 0.1) from day 0 and day 4 post infection calculated by the ΔΔCt method (determined as in part A). C) IFNβ mRNA expression of LH86 cells electroporated with in vitro transcribed HCV/JFH-1 or its non-replicating counterpart HCV/JFH-GND (day 0 is the day of the electroporation). Expression was calculated as described for part A.
Figure 2. Both TLRs and RIG-I prevent viral replication and are needed for the induction of IFN.
A) IFNβ mRNA expression at day 4 post-infection in LH86 cells after transfecting with a control siRNA, or siRNA against TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. No virus indicates cells were cultured with the same volume in uninfected Huh7. 5 supernatant, HCV is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1). B) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells expressing TLR3 or TLR7. Expression was calculated by the ΔΔCt method as described in part A and bars represent the SEM of three separate experiments. C) Viral replication in Huh7.5 cells expressing TLR3 or TLR7 at day 7 after infection. HCV copy numbers were calculated by real time RT-PCR run with an HCV standard curve. D) IFNβ mRNA expression at day 4 post-infection in LH86 cells with silenced RIG-I. Methodology as described in part A. E) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells transfected with RIG-I. Methodology as described in part A. F) Viral replication in Huh7.5 cells transfected with RIG-I after 7 days of culture. Methodology as described in part C.
Figure 3. RIG-I is linked to the expression of TRAIL receptors DR4 and DR5.
A) TRAIL mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) DR4 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in A. C) DR5 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in part A. D) TRAIL mRNA expression 4 days after infection of LH86 cells silenced for RIG-I, TLR3 or TLR7 calculated as described for part A. E) DR4 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. F) DR5 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. G) Phase view (100X magnification) of transfected cells 7 days after infection. Top row are LH86 cells and bottom row Huh7.5 cells. The labels note what each cell was transfected with.
Figure 4. A strong initial IFN response is induced an IRF-3 response by TLR3 but is not enough to clear HCV.
A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.
Figure 5. The expression of TLR3 and RIG-I is affected by intact virion proteins in hepatocytes
A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in figure 1C). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.
Figure 6. Envelope proteins affect the response to non-HCV responses through RIG-I receptor but only temporarily through TLR3.
A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.
Figure 7. Schematics of the initial interaction of HCV in hepatocytes.
Virus binds and gets in the cells were nucleic acid or replication induces the host cells innate immunity through the induction of apoptosis (mediated by RIG-I) and IFNβ (through TLR3 engagement). While the replication of the virus leads to the development of viral proteins like NS34A that interact downstream of these effects (red arrow line) there is a possible earlier evasion strategy performed by virion proteins that lead to the down regulation of RIG-I and TLR3 preventing apoptosis and the induction of IFNβ. Apoptosis is particularly prevented by down regulating TRAIL receptors, DR4 and DR5, and IFNβ by preventing NF-κB pathways such as the one that induces IP-10. Together all of these factors help the cell survive and the virus to persist in the host's hepatocytes.
References
- Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med. 2000;132:296–305. - PubMed
- Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis. 2005;5:558–567. - PubMed
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