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Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells.

Eksioglu EA, Zhu H, Bayouth L, Bess J, Liu HY, Nelson DR, Liu C - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, United States of America.

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.

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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.
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pone-0021186-g004: 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.

Mentions: To determine the functional role of TLR3 and TLR7, we examined the relationship between viral replication and the IFN response in a long-term culture system. The cells were infected with HCV-JFH1 virus and samples were collected every 2–3 days up to 75 days. Viral RNA replication levels were determined by real-time RT-PCR analysis. As shown in Figure 4, both TLR3 and TLR7 had a profound effect on viral replication throughout the time of analysis. The cells with both TLR3 and TLR7 suppressed HCV replication, while TLR3 has the most profound effect (Figure 4A). To test whether this antiviral effect correlates with an IFN response, we quantified IFNβ expression by real time RT-PCR. As shown in Figure 4B, over-expressing TLR3 in Huh7.5 cells induced a strong IFN production only in the first week, while TLR7 induced a very low level of IFN. TLR3 has at least two potential signaling pathways: NF-κB or IRF-3 cascades. To determine which pathway is operative under these conditions, we measured the expression of cytokines specific for each pathway (RANTES which is IRF-3 induced and IP-10 which is NF-κB induced) [26], [27], [28]. We observed that IP-10 was down-regulated in Huh7.5 TLR3 cells throughout the culture while RANTES was induced and sustained throughout the culture period indicating that the down regulation of viral replication correlates with the induction of the IRF-3 pathway without apparent NF-κB activation (Figure 4C).


Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells.

Eksioglu EA, Zhu H, Bayouth L, Bess J, Liu HY, Nelson DR, Liu C - PLoS ONE (2011)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117876&req=5

pone-0021186-g004: 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.
Mentions: To determine the functional role of TLR3 and TLR7, we examined the relationship between viral replication and the IFN response in a long-term culture system. The cells were infected with HCV-JFH1 virus and samples were collected every 2–3 days up to 75 days. Viral RNA replication levels were determined by real-time RT-PCR analysis. As shown in Figure 4, both TLR3 and TLR7 had a profound effect on viral replication throughout the time of analysis. The cells with both TLR3 and TLR7 suppressed HCV replication, while TLR3 has the most profound effect (Figure 4A). To test whether this antiviral effect correlates with an IFN response, we quantified IFNβ expression by real time RT-PCR. As shown in Figure 4B, over-expressing TLR3 in Huh7.5 cells induced a strong IFN production only in the first week, while TLR7 induced a very low level of IFN. TLR3 has at least two potential signaling pathways: NF-κB or IRF-3 cascades. To determine which pathway is operative under these conditions, we measured the expression of cytokines specific for each pathway (RANTES which is IRF-3 induced and IP-10 which is NF-κB induced) [26], [27], [28]. We observed that IP-10 was down-regulated in Huh7.5 TLR3 cells throughout the culture while RANTES was induced and sustained throughout the culture period indicating that the down regulation of viral replication correlates with the induction of the IRF-3 pathway without apparent NF-κB activation (Figure 4C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, United States of America.

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.

Show MeSH
Related in: MedlinePlus