Ethanol facilitates hepatitis C virus replication via up-regulation of GW182 and heat shock protein 90 in human hepatoma cells

Abstract

Alcohol use and hepatitis C virus (HCV) infection synergize to cause liver damage, and microRNA-122 (miR-122) appears to play a key role in this process. Argonaute 2 (Ago2), a key component of the RNA-induced silencing complex (RISC), has been shown to be important in modulating miR-122 function during HCV infection. However, GW182, a critical component of processing bodies (GW bodies) that is recruited by Ago2 to target messenger RNA (mRNA), has not been assessed in HCV infection. To characterize the role of GW182 in the pathogenesis of HCV infection, we determined its transcription and protein expression in an HCV J6/JFH1 culture system. Transcript and protein levels of GW182 as well as HCV RNA and protein expression increased with alcohol exposure. Specific silencing of mRNA expression by small interfering RNA against GW182 significantly decreased HCV RNA and protein expression. Overexpression of GW182 significantly increased HCV RNA and protein expression in HCV J6/JFH1 infected Huh7.5 cells. Furthermore, GW182 colocalized and coimmunoprecipitated with heat shock protein 90 (HSP90), which increased upon alcohol exposure with and without HCV infection and enhanced HCV gene expression. The use of an HSP90 inhibitor or knockdown of HSP90 decreased GW182 and miR-122 expression and significantly reduced HCV replication.

Conclusion: Overall, our results suggest that GW182 protein that is linked to miR-122 biogenesis and HSP90, which has been shown to stabilize the RISC, are novel host proteins that regulate HCV infection during alcohol abuse.

Figure 1. Acute ethanol treatment modulated the expression of GW182

(A–E) The expression of GW182 mRNA, GW182 protein, HCV RNA, HCV NS3 and GW-bodies was assessed as indicated. (A) GW182 mRNA (B) GW182 protein (C) HCV RNA (D) HCV NS3 protein were evaluated 24hr after ethanol exposure in J6/JFH1-infected and non-infected Huh-7.5 cells. (E) Huh-7.5 cells were treated with ethanol (0mM or 25mM). The expression of GW-bodies with GW182 as marker was examined by fluorescence microscopy after 24hr ethanol exposure. Fixed cells were stained with a rabbit polyclonal antibody to GW182 (red). GW182 foci levels were determined for at least 35 cells chosen randomly from different fields using the Olympus BX51 fluorescence microscope for imaging and the Nixon Nikon NIS-Element BR 3.10 software for acquisition and the NIH ImageJ 1.46 software quantification. Results presented are representative of 3–4 independent repeat experiments and data expressed as SEM, p< 0.05 were considered statistically significant (by two-tailed Student’s t test. Multiplicity of infection (MOI) of 1 was used for all infections.

Figure 2. GW182 is essential for HCV replication

J6/JFH1-infected or uninfected Huh-7.5 cells with and without ethanol exposure were transfected with GW182 siRNA (50nM) or control siRNA (50nM). 48hr after transfection, (A) GW182 mRNA (RT-qPCR), (B) intracellular HCV RNA (RT-qPCR), (C) HCV NS3 protein (western blot) expression and (D) the miR-122 relative expression was quantified. (E) Over-expression of GW182 confirmed with an anti-HA antibody in J6/JFH1-infected cells and NS3HCV protein expression compared to controls with and without alcohol exposure. (F) miR-122 expression determined with and without alcohol exposure following GW182 over expression. Results presented are representative of 3–4 independent repeat experiments and data expressed as SEM, p< 0.05 were considered statistically significant (by two-tailed Student’s t test). Multiplicity of infection (MOI) of 1 was used for all infections.

Figure 3. GW182 co-localized and co-immunoprecipitated with viral proteins and HSP 90

(A) The co-localization of GW182 (stained with rabbit polyclonal antibody to GW182 (Red) and HCV protein (Green) were evaluated by fluorescence microscopy in J6/JFH1-infected Huh-7.5 cells. (B & C) Co-immunoprecipitation of GW182 with HSP 90 and viral proteins was evaluated in J6/JFH1-infected Huh 7.5 cells. (D) The co-localization of GW182 (stained with a mouse monoclonal antibody to GW182 (Red) and HSP 90 protein (Green) were evaluated by confocal microscopy in J6/JFH1-infected and uninfected Huh-7.5 cells. (E) Co-immunoprecipitation of HSP 90 with GW182 was evaluated in J6/JFH1-infected and uninfected Huh 7.5 cells. Results presented are representative of 3–4 independent repeat experiments with at least 10 fields sequential analyzed for each microscopy slide to minimize spectral bleed-through artifacts. Multiplicity of infection (MOI) of 1 was used for IP infections and MOI of 0.5 for microscopy slides.

Figure 4. HSP90 co-localized and co-immunoprecipitated with HCV proteins

(A) The co-localization of HSP90 (Red) and HCV proteins (Green) were evaluated by fluorescence microscopy and (B) co-immunoprecipitation in J6/JFH1-infected Huh-7.5 cells. (A) Representative slides with at least 10 fields sequential analyzed for each microscopy slide to minimize spectral bleed-through artifacts. Results presented are representative of 3 independent repeat experiments. Multiplicity of infection (MOI) of 1 was used for IP infections and MOI of 0.5 for microscopy slides.

Figure 5. HSP90 has a mechanistic role in regulation of HCV replication and miR-122

(A) Huh-7.5 and J6/JFH1-infected Huh-7.5 cells were treated with ethanol, 17-DMAG or not for 24hr and HCV NS3 and HSP90 protein analyzed. (B) Huh-7.5 and J6/JFH1-infected Huh-7.5 cells with and without ethanol exposure were transfected with HSP90 (50nM), or control siRNA; HCV NS3 protein (B), HSP90 RNA (C), HSP90 protein (B), miR-122 (E) and GW182 RNA (F) was evaluated after 24hrs. (D) Huh-7.5 and J6/JFH1-infected Huh-7.5 cells were treated with ethanol, 17-DMAG or not for 24hr and miR-122 expression determined. (C, D, E&F) Data expressed as SEM, p< 0.05 were considered statistically significant (by two-tailed Student’s t test).

Figure 6. Acute ethanol treatment induces miR-122 and activates hepatitis C virus replication

(A&B) J6/JFH-infected and uninfected Huh-7.5 cells were transfected with a miR-122 inhibitor (50nM) or negative control (50nM) prior to ethanol treatment (0mM or 25mM). Intracellular HCV RNA (A) and NS3 protein (B) was quantified (RT-qPCR) 24hr after ethanol exposure. Results presented are representative of 3 independent repeat experiments and data expressed as SEM, p< 0.05 were considered statistically significant (by two-tailed Student’s t test). Multiplicity of infection (MOI) of 1 was used for these infections.