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. 2013 May 31;8(5):e64932.
doi: 10.1371/journal.pone.0064932. Print 2013.

2-octynoic acid inhibits hepatitis C virus infection through activation of AMP-activated protein kinase

Darong Yang  1 , Binbin Xue, Xiaohong Wang, Xiaoyan Yu, Nianli Liu, Yimin Gao, Chen Liu, Haizhen Zhu
Affiliations

2-octynoic acid inhibits hepatitis C virus infection through activation of AMP-activated protein kinase

Darong Yang et al. PLoS One. .

Abstract

Many chronic hepatitis C virus (HCV)-infected patients with current therapy do not clear the virus. It is necessary to find novel treatments. The effect of 2-octynoic acid (2-OA) on HCV infection in human hepatocytes was examined. The mechanism of 2-OA antiviral activity was explored. Our data showed that 2-OA abrogated lipid accumulation in HCV replicon cells and virus-infected hepatocytes. It suppressed HCV RNA replication and infectious virus production with no cytotoxicity to the host cells. 2-OA did not affect hepatitis B virus replication in HepG2.2.15 cells derived from HepG2 cells transfected with full genome of HBV. Further study demonstrated that 2-OA activated AMP-activated protein kinase (AMPK) and inhibited acetyl-CoA carboxylase in viral-infected cells. Compound C, a specific inhibitor of AMPK, inhibited AMPK activity and reversed the reduction of intracellular lipid accumulation and the antiviral effect of 2-OA. Knockdown of AMPK expression by RNA interference abolished the activation of AMPK by 2-OA and blocked 2-OA antiviral activity. Interestingly, 2-OA induced interferon-stimulated genes (ISGs) and inhibited microRNA-122 (miR-122) expression in virus-infected hepatocytes. MiR-122 overexpression reversed the antiviral effect of 2-OA. Furthermore, knockdown of AMPK expression reversed both the induction of ISGs and suppression of miR-122 by 2-OA, implying that activated AMPK induces the intracellular innate response through the induction of ISGs and inhibiting miR-122 expression. 2-OA inhibits HCV infection through regulation of innate immune response by activated AMPK. These findings reveal a novel mechanism by which active AMPK inhibits HCV infection. 2-OA and its derivatives hold promise for novel drug development for chronic hepatitis C.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of 2-OA on lipid accumulation in HCV replicon cells and infectious cell culture system.
(A) Cellular lipid was evaluated in FCA1, FL-Neo cells and HCV-infected Huh7.5 cells (Huh7.5 HCVcc) in the presence of 100 µM 2-OA for 48 hours. Cells were stained for lipid content with BODIPY dye (green). DAPI was used for nuclear counterstaining (blue). Identical settings were maintained for image capture. Representative images are shown (upper). For quantification of lipid abundance, images were captured and analyzed using image analysis software. Values were normalized to control BODIPY levels (lower). (B) The effect of 2-OA on viability of FCA1, FL-Neo and Huh7.5 HCVcc cells was measured by MTS assay. The data were normalized with untreated cells.
Figure 2
Figure 2. 2-OA inhibits HCV RNA replication in replicon cells.
(A/B) FCA1 (A) and FL-Neo (B) were incubated with 10 µM or 100 µM 2-OA for 48, 72 hours. HCV RNA was detected with real-time PCR and normalized with internal control GAPDH. EC50 of 2-OA is obtained from Figure 2A (Lower). (C) FCA1 and FL-Neo were incubated with 100 µM 2-OA for 48 hours and immunostained with mouse monoclonal anti-NS5A antibody (red). DAPI was used for nuclear counterstaining (blue). Identical settings were maintained for image capture. Representative images are shown. (D) FCA1 cells were treated with 2-OA for 48 hours. NS5A protein was detected with western blot (upper) and quantified by densitometry in comparison with β-actin (lower). (E) 2-OA did not affect hepatitis B viral DNA replication. HepG2.2.15 cells were inoculated with different doses of 2-OA for 72 hours. Intracellular HBV DNA was detected with real-time PCR and normalized with GAPDH.
Figure 3
Figure 3. 2-OA inhibits HCV infection in infectious cell culture system.
(A)/(B) 2-OA inhibits HCV replication in JFH1 RNA-transfected Huh7.5 cells. Huh7.5 cells were transfected with in vitro transcribed JFH1 RNA. (A) The cells were treated with 10 µM and 100 µM 2-OA for 48 and 72 hours. HCV RNA was detected with real-time PCR and normalized with GAPDH. (B) The cells were treated with 100 µM 2-OA for 48 hours and immunostained with mouse monoclonal anti-NS5A antibody (red). DAPI was used for nuclear counterstaining (blue). Identical settings were maintained for image capture. Representative images are shown. (C) Huh7.5 cells were infected by JFH1 virus at MOI of 0.1 in the presence of different doses of 2-OA for 72 hours. Intracellular HCV RNA was analyzed by real-time PCR and displayed as genome equivalents (GE)/µg total cellular RNA. (D) Huh7.5 cells were infected by JFH1 virus at MOI of 0.1 for 2 days and treated by 2-OA for 72 hours. NS5A protein was detected with western blot analysis (upper) and quantified by densitometry in comparison with β-actin (lower). (E) Huh7.5 cells were infected with JFH1 virus at MOI of 0.1 and treated by 100 µM 2-OA for 72 hours. The extracellular or intracellular virus was titred. The infectivity titers in the supernatant or cell lysates were normalized to that of control from three independent experiments.
Figure 4
Figure 4. 2-OA antiviral effect is associated with AMPK activation.
(A)/(B) 2-OA activates AMPK (A) and phosphorylates ACC (B) in replicon cells. Huh7 and FCA1 cells were treated with 100 µM 2-OA for 10 minutes. pT172 of AMPK and pSer79 of ACC were detected by western blot respectively. Levels of phosphorylated AMPK or ACC were quantified by densitometry in comparison with β-actin (lower). (C) Compound C (CC) blocks activation of AMPK by 2-OA. FCA1 cells were treated by 100 µM 2-OA and 10 µM compound C for 10 minutes. pT172 of AMPK was detected by western blot and quantified by densitometry in comparison with β-actin (lower). (D) Compound C inhibits 2-OA antiviral activity. FCA1 cells were treated with 100 µM 2-OA and 2.5 µM compound C for 48 hours. Cells were stained for lipid content with BODIPY dye (green) after NS5A labeling by immunofluorescence (red). DAPI was used for nuclear counterstaining (blue). Identical settings were maintained for image capture (left). Representative confocal images are shown. NS5A and lipid levels were quantified by densitometry and normalized to control cells (right). (E)/(F) Knockdown of AMPK abolishes 2-OA antiviral activity (E) and abrogates the suppression of infectious virus production by 2-OA (F). AMPK siRNA was delivered into HCV-infected cells. The cells were treated by 100 µM 2-OA for 72 hours. (E) AMPK protein or NS5A was detected by western blot (upper). NS5A levels were quantified by densitometry in comparison with β-actin (lower). (F) The extracellular or intracellular virus was titred. The infectivity titers in the supernatant or cell lysates are were normalized to that of control cells from three independent experiments.
Figure 5
Figure 5. 2-OA induces ISGs and inhibits the expression of miR-122 through activated AMPK.
(A)/(B) 2-OA induces G1P3 (A) and IRF-1 (B) in HCV-infected Huh7.5. Virus-infected cells were incubated with 100 µM 2-OA for different time periods. G1P3 (A) or IRF-1 (B) RNA was detected with real-time PCR. The expression of G1P3 or IRF-1 was normalized with GAPDH. The data represented the means of 3 different experiments. (C) 2-OA suppresses miR-122 expression in HCV-infected Huh7.5 cells. Virus-infected hepatocytes were treated with 100 µM 2-OA for different time periods. MiR-122 was detected with real-time PCR. The expression of miR-122 was normalized with GAPDH. (D)/(E) Knockdown of AMPK by AMPK siRNA abrogates the induction of G1P3 and IRF-1 by 2-OA. Virus-infected Huh7.5 cells pretransfected by AMPK siRNA or control siRNA were incubated with 100 µM 2-OA for 2 hours. G1P3 (D) or IRF-1 (E) RNA was detected with real-time PCR. The expression of G1P3 or IRF-1 was normalized with GAPDH. (F) Knockdown of AMPK by AMPK siRNA reverses the inhibition of miR-122 by 2-OA. HCV-infected Huh7.5 cells pretransfected by AMPK siRNA or control siRNA were treated with 100 µM 2-OA for 48 hours. MiR-122 was detected with real-time PCR. The expression of miR-122 was normalized with GAPDH. (G) MiR-122 overexpression reversed the antiviral effect of 2-OA. MiR-122 was overexpressed in HCV-infected cells and the cells were treated by 100 µM 2-OA for 48 hours. Total cellular RNA was isolated from the cells. HCV RNA was detected by real-time PCR analysis and normalized with GAPDH.
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Grants and funding

This work was supported by National Science and Technology Major Project of the Ministry of Science and Technology of China (2009ZX10004-312), National Natural Science Foundation of China (81271885), and Program for New Century Excellent Talents in University (NCET-09-0339) to HZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.