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EPSTI1 Is Involved in IL-28A-Mediated Inhibition of HCV Infection.

Meng X, Yang D, Yu R, Zhu H - Mediators Inflamm. (2015)

Bottom Line: In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release.EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity.EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-β, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine of College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China.

ABSTRACT
It has been reported that IFN-λs inhibit HCV replication in vitro. But the mechanisms of how IL-28A conducts antiviral activity and the functions of IL-28A-induced ISGs (IFN-stimulated genes) are not fully understood. In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release. IL-28A and IFN-α synergistically inhibit virus replication. EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity. Furthermore, forced expression of EPSTI1 effectively inhibits HCV replication in the absence of interferon treatment, and knockdown of EPSTI1 contributes to viral enhancement. EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-β, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.

No MeSH data available.


Related in: MedlinePlus

IL-28A suppresses HCV replication. Huh7.5 (a) and HLCZ01 (c) cells were infected with HCV (MOI = 0.1) for 6 h at 37°C. The medium was then removed and cells were incubated with IL-28A for 48 or 72 h at 37°C. (b) HCV-infected Huh7.5 cells were incubated with different doses of IL-28A for 72 h. (d) FL cells were incubated with IL-28A for 48 or 72 h. ((a), (b), (c), and (d)) Intracellular viral RNA was detected by real-time PCR. The results are the average of three independent experiments performed in triplicate. (e) Huh7.5 cells were treated as described in part (a). NS5A and core proteins were detected with western blot. (f) Huh7.5 cells were pretreated with or without IL-28A for 12 h. The medium was then removed, and cells were inoculated with HCV (MOI = 0.1) for 6 h at 37°C. Intracellular viral RNA was detected by real-time PCR.
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fig1: IL-28A suppresses HCV replication. Huh7.5 (a) and HLCZ01 (c) cells were infected with HCV (MOI = 0.1) for 6 h at 37°C. The medium was then removed and cells were incubated with IL-28A for 48 or 72 h at 37°C. (b) HCV-infected Huh7.5 cells were incubated with different doses of IL-28A for 72 h. (d) FL cells were incubated with IL-28A for 48 or 72 h. ((a), (b), (c), and (d)) Intracellular viral RNA was detected by real-time PCR. The results are the average of three independent experiments performed in triplicate. (e) Huh7.5 cells were treated as described in part (a). NS5A and core proteins were detected with western blot. (f) Huh7.5 cells were pretreated with or without IL-28A for 12 h. The medium was then removed, and cells were inoculated with HCV (MOI = 0.1) for 6 h at 37°C. Intracellular viral RNA was detected by real-time PCR.

Mentions: In previous study, we have investigated the anti-HCV activity of IL-28A in HCV subgenomic replicon cell line [12]. The recent development of infectious HCV cell culture system, a genotype 2a patient isolate JFH1, facilitates the study of anti-HCV drugs. Here, we assessed the antiviral activity of IL-28A in infectious cell culture system and a HCV genotype 1b full-length replicon cell line (FL-neo). Huh7.5 cells were inoculated with purified JFH1 at MOI of 0.1 for 6 hours. After the removal of inoculum, the cells were cultured with the fresh media containing different doses of IL-28A. Viral RNA replication in Huh7.5 cells was suppressed by IL-28A in both dose- and time-dependent manners (Figure 1(a)). The EC50 of IL-28A for inhibition of HCV RNA replication in Huh7.5 cells was 10.31 ng/mL (Figure 1(b)). We also assessed the antiviral activity of IL-28A in our newly developed hepatoma cell line HLCZ01 with HCV infection [24]. IL-28A inhibited HCV RNA replication in viral-infected HLCZ01 cells in both dose- and time-dependent manners (Figure 1(c)). Moreover, IL-28A also repressed HCV RNA replication in FL-neo cells (Figure 1(d)), suggesting that IL-28A may act as an antiviral drug for multigenotypes of HCV. To further confirm the inhibition of IL-28A on HCV replication, we detected viral proteins in the cells. As shown in Figure 1(e), NS5A and core proteins were significantly reduced in HCV-infected cells with IL-28A treatment. To investigate whether IL-28A has effect on HCV entry, we pretreated Huh7.5 cells with IL-28A for 12 hours followed by HCV infection for 6 hours. The result indicated that IL-28A treatment does not affect the efficiency of HCV entry (Figure 1(f)). These data suggested that IL-28A inhibits HCV replication without affecting HCV entry.


EPSTI1 Is Involved in IL-28A-Mediated Inhibition of HCV Infection.

Meng X, Yang D, Yu R, Zhu H - Mediators Inflamm. (2015)

IL-28A suppresses HCV replication. Huh7.5 (a) and HLCZ01 (c) cells were infected with HCV (MOI = 0.1) for 6 h at 37°C. The medium was then removed and cells were incubated with IL-28A for 48 or 72 h at 37°C. (b) HCV-infected Huh7.5 cells were incubated with different doses of IL-28A for 72 h. (d) FL cells were incubated with IL-28A for 48 or 72 h. ((a), (b), (c), and (d)) Intracellular viral RNA was detected by real-time PCR. The results are the average of three independent experiments performed in triplicate. (e) Huh7.5 cells were treated as described in part (a). NS5A and core proteins were detected with western blot. (f) Huh7.5 cells were pretreated with or without IL-28A for 12 h. The medium was then removed, and cells were inoculated with HCV (MOI = 0.1) for 6 h at 37°C. Intracellular viral RNA was detected by real-time PCR.
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Related In: Results  -  Collection

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fig1: IL-28A suppresses HCV replication. Huh7.5 (a) and HLCZ01 (c) cells were infected with HCV (MOI = 0.1) for 6 h at 37°C. The medium was then removed and cells were incubated with IL-28A for 48 or 72 h at 37°C. (b) HCV-infected Huh7.5 cells were incubated with different doses of IL-28A for 72 h. (d) FL cells were incubated with IL-28A for 48 or 72 h. ((a), (b), (c), and (d)) Intracellular viral RNA was detected by real-time PCR. The results are the average of three independent experiments performed in triplicate. (e) Huh7.5 cells were treated as described in part (a). NS5A and core proteins were detected with western blot. (f) Huh7.5 cells were pretreated with or without IL-28A for 12 h. The medium was then removed, and cells were inoculated with HCV (MOI = 0.1) for 6 h at 37°C. Intracellular viral RNA was detected by real-time PCR.
Mentions: In previous study, we have investigated the anti-HCV activity of IL-28A in HCV subgenomic replicon cell line [12]. The recent development of infectious HCV cell culture system, a genotype 2a patient isolate JFH1, facilitates the study of anti-HCV drugs. Here, we assessed the antiviral activity of IL-28A in infectious cell culture system and a HCV genotype 1b full-length replicon cell line (FL-neo). Huh7.5 cells were inoculated with purified JFH1 at MOI of 0.1 for 6 hours. After the removal of inoculum, the cells were cultured with the fresh media containing different doses of IL-28A. Viral RNA replication in Huh7.5 cells was suppressed by IL-28A in both dose- and time-dependent manners (Figure 1(a)). The EC50 of IL-28A for inhibition of HCV RNA replication in Huh7.5 cells was 10.31 ng/mL (Figure 1(b)). We also assessed the antiviral activity of IL-28A in our newly developed hepatoma cell line HLCZ01 with HCV infection [24]. IL-28A inhibited HCV RNA replication in viral-infected HLCZ01 cells in both dose- and time-dependent manners (Figure 1(c)). Moreover, IL-28A also repressed HCV RNA replication in FL-neo cells (Figure 1(d)), suggesting that IL-28A may act as an antiviral drug for multigenotypes of HCV. To further confirm the inhibition of IL-28A on HCV replication, we detected viral proteins in the cells. As shown in Figure 1(e), NS5A and core proteins were significantly reduced in HCV-infected cells with IL-28A treatment. To investigate whether IL-28A has effect on HCV entry, we pretreated Huh7.5 cells with IL-28A for 12 hours followed by HCV infection for 6 hours. The result indicated that IL-28A treatment does not affect the efficiency of HCV entry (Figure 1(f)). These data suggested that IL-28A inhibits HCV replication without affecting HCV entry.

Bottom Line: In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release.EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity.EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-β, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine of College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China.

ABSTRACT
It has been reported that IFN-λs inhibit HCV replication in vitro. But the mechanisms of how IL-28A conducts antiviral activity and the functions of IL-28A-induced ISGs (IFN-stimulated genes) are not fully understood. In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release. IL-28A and IFN-α synergistically inhibit virus replication. EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity. Furthermore, forced expression of EPSTI1 effectively inhibits HCV replication in the absence of interferon treatment, and knockdown of EPSTI1 contributes to viral enhancement. EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-β, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.

No MeSH data available.


Related in: MedlinePlus