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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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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.
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pone-0021186-g006: 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.

Mentions: The observations just described combined with our initial evidence (Figure 1A) demonstrates that non-replicating virus can down-regulate IFN which indicates there is likely a role by virion proteins in immune-evasion. To understand how these HCV proteins can impact the innate immune system we created stable cell lines with LH86 expressing three viral proteins: core, envelope (E1/E2) and NS3/4A (already known to affect IFN expression) [30]. These stable cell lines were infected with HCV at an MOI of 0.1 as before and the IFN response was measured. As expected, LH86 cells transfected with NS3/4A had a reduced IFN peak at day 4 as compared to control cells (Figure 6A). In contrast, LH86 cells transfected with core or envelope did not induce IFN. Core-transfected cells did have an earlier peak (not shown) but by day 4 IFN was no different than uninfected cells. Conversely, LH86 cells that harbored the envelope proteins did not express IFN at any time point during a 7 day culture and at day 4 the basal IFN expression on those cells was down regulated as well. Furthermore, these transfected cells were able to down-regulate the levels of TLR3 (Figure 6B) and RIG-I (Figure 6C) but only the envelope-transfected cells maintained the down-regulation of RIG-I after HCV infection.


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)

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.
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pone-0021186-g006: 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.
Mentions: The observations just described combined with our initial evidence (Figure 1A) demonstrates that non-replicating virus can down-regulate IFN which indicates there is likely a role by virion proteins in immune-evasion. To understand how these HCV proteins can impact the innate immune system we created stable cell lines with LH86 expressing three viral proteins: core, envelope (E1/E2) and NS3/4A (already known to affect IFN expression) [30]. These stable cell lines were infected with HCV at an MOI of 0.1 as before and the IFN response was measured. As expected, LH86 cells transfected with NS3/4A had a reduced IFN peak at day 4 as compared to control cells (Figure 6A). In contrast, LH86 cells transfected with core or envelope did not induce IFN. Core-transfected cells did have an earlier peak (not shown) but by day 4 IFN was no different than uninfected cells. Conversely, LH86 cells that harbored the envelope proteins did not express IFN at any time point during a 7 day culture and at day 4 the basal IFN expression on those cells was down regulated as well. Furthermore, these transfected cells were able to down-regulate the levels of TLR3 (Figure 6B) and RIG-I (Figure 6C) but only the envelope-transfected cells maintained the down-regulation of RIG-I after HCV infection.

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