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The Ribonuclease L-Dependent Antiviral Roles of Human 2€²,5€²

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The Ribonuclease L-Dependent Antiviral Roles of Human 2€²,5€² View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 587 (2013) 156–164 journal homepage: www.FEBSLetters.org The ribonuclease L-dependent antiviral roles of human 20,50-oligoadenylate synthetase family members against hepatitis C virus ⇑ Young-Chan Kwon a, Ju-Il Kang a, Soon B. Hwang b, Byung-Yoon Ahn a, a School of Life Sciences and Biotechnology, Korea University, Anam-dong 5-1, Seoul 136-701, Republic of Korea b Ilsong Institute of Life Science, Hallym University, Gwanyang-dong 1605-4, Anyang 431-060, Republic of Korea article info abstract Article history: The latent ribonuclease RNase L and the interferon-inducible 20,50-oligoadenylate synthetase (OAS) Received 16 October 2012 have been implicated in the antiviral response against hepatitis C virus (HCV). However, the specific Accepted 9 November 2012 roles of these enzymes against HCV have not been fully elucidated. In this study, a scarce endoge- Available online 26 November 2012 nous expression and RNA degrading activity of RNase L in human hepatoma Huh7 cells enabled us to demonstrate the antiviral activity of RNase L against HCV replication through the transient Edited by Hans-Dieter Klenk expression of the enzyme. The antiviral potential of specific members of the OAS family was further examined through overexpression and RNA interference approaches. Our data suggested that Keywords: among the members of the OAS family, OAS1 p46 and OAS3 p100 mediate the RNase L-dependent RNase L, 20,50-Oligoadenylate synthetase antiviral activity against HCV. Hepatitis C virus Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Antiviral protein 1. Introduction often lead to chronic infection, cirrhosis and hepatocellular carci- noma. It is estimated that 170 million individuals around the world RNase L is a latent ribonuclease that is expressed constitutively are currently infected with HCV. Type I IFN in combination with and ubiquitously in almost all mammalian cells. The binding of ribavirin and one of the two recently approved protease inhibitors 20,50-linked oligoadenylate (2–5A) to the N-terminal ankyrin re- is the current standard therapy for chronic hepatitis C [5]. peats of RNase L induces the dimerization of the enzyme and the RNase L and OAS have been implicated in the antiviral response activation of its endonuclease activity. Activated RNase L cleaves against HCV. The two proteins were found to be abundant in the viral and cellular RNAs at single-stranded regions, resulting in the liver tissues of patients with chronic hepatitis C, and they co-local- inhibition of the translation and replication of viral RNA [1]. In addi- ized with HCV NS5A protein, suggesting that they are likely en- tion to the direct antiviral effect of RNase L, an indirect antiviral role gaged in antagonizing interactions with HCV [6]. It has been for this enzyme has been suggested; in particular, it has been found that OAS transcripts are induced in the liver of HCV patients hypothesized that the small RNA fragments that are generated by and model animals [7,8]. HCV patients undergoing IFN therapy RNase L can act as a signal for interferon (IFN) production [2]. demonstrated an elevated level of OAS in sera; this increase in The structurally unique 2–5A, which is a specific activator of OAS was correlated with predicted virological responses in one RNase L, is synthesized through the oligomerization of ATP, which study [9], but they were uncorrelated in another study [10]. is catalyzed by the 20,50-oligoadenylate synthetases (OASs). Despite the strong implications that RNase L and OAS play a role Although the transcription of the OAS gene is induced by IFNs, in the response to HCV infection, their specific roles against HCV OAS proteins are translated as latent enzymes before they are acti- have not yet been elucidated. The cleavage of the HCV genome vated by the binding of dsRNA; this dsRNA is likely of viral origin. RNA by RNase L has been demonstrated in vitro [11]. The RNase Thus, OAS in combination with RNase L constitutes an essential L-mediated cleavage products of transfected HCV RNA triggered antiviral pathway that is induced in response to IFN [3]. IFN producing signals, suggesting an indirect antiviral role of RNase Hepatitis C virus (HCV) is a hepatotrophic member of the flavi- L against HCV [6]. Recently, RNase L was identified along with sev- viruses that possesses a positive-strand RNA genome [4]. HCV-in- eral cellular IFN-stimulated proteins as antiviral proteins that re- fected individuals typically develop persistent infections that strict HCV replicons through an RNAi-based knock-down screening approach [12]. In this study, we demonstrated the anti- viral activity of RNase L against HCV replication in human hepa- ⇑ Corresponding author. Fax: +82 2 923 9923. toma Huh7 cells through transient expression of the enzyme. Our E-mail address: [email protected] (B.-Y. Ahn). data further suggested that two members of the OAS family, 0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.11.010 Y.-C. Kwon et al. / FEBS Letters 587 (2013) 156–164 157 OAS1 p46 and OAS3 p100, mediate the RNase L-dependent antivi- 2.4. The immunoblotting and immunofluorescence assays ral activity against HCV infection. For immunoblotting, cells were lysed in RIPA buffer (10 mM 2. Materials and methods Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.1% SDS, 0.25% sodium deoxycholate, and 1 mM PMSF). The pro- 2.1. Cells and viruses teins were separated in a denaturating polyacrylamide gel and transferred onto nitrocellulose membranes (Hybond ECL; GE Human hepatoma Huh7 and Huh7.5 cells were grown in Dul- Healthcare). The blots were incubated with primary and secondary becco’s modified Eagle’s medium (DMEM) that had been supple- antibodies that had been diluted in TBST buffer (10 mM Tris–HCl mented with 50 lg/ml of gentamicin and 5% fetal bovine serum [pH 7.6], 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk (Hyclone) at 37 °C in an atmosphere containing 5% CO2. HepG2 and were visualized with PicoWest SuperSignal ECL reagents cells were cultured in the aforementioned medium that had been (Pierce). Polyclonal antibodies to NS5A and MxA were raised in supplemented with 10% FBS. Human lung epithelial carcinoma accordance with previously described procedures [15]. The anti- A549 cells were maintained in RPMI 1640 medium that had been RNase L antibody that was used was purchased from Invitrogen. supplemented with 10% FBS. HCV replicon cells were prepared The anti-tubulin antibody that was used was purchased from Ap- by the electroporation of RNA from the genotype 2a HCV strains plied Biological Materials. The anti-Flag and anti-HA antibodies of JFH-1 [13], J6/JFH-1 [14] or SGR/Luc-JFH1 with a Gene Pulser II were purchased from Sigma, and the anti-core antibody was pur- RF module (Bio-Rad) in accordance with previously described pro- chased from Pierce. cedures [15]. The viral RNA genome was synthesized in vitro from For immunostaining, cells were fixed with 3.7% paraformalde- appropriate templates with the MEGAscript T7 Kit (Ambion). Virus hyde in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, titer in the culture supernatant was determined in the focus-form- 2 mM MgSO4, pH 7.0), permeabilized for 10 min in PBS with 0.5% ing assay in accordance with previously characterized procedures Triton X-100, and incubated for 10 min in blocking buffer (PBS con- [16]. As described below, viral replication was assessed by immu- taining 1% BSA and 0.1% Tween 20). The cells were incubated for noblotting, northern blotting or RT-PCR analyses. The luciferase 1 h at room temperature with a 1:1,000 dilution of primary anti- activity for SGR/Luc-JFH1 was measured with the Luciferase Assay bodies in blocking buffer, washed with PBS and incubated for 1 h System (Promega). The recombinant Newcastle disease virus at room temperature in blocking buffer with a 1:1,000 dilution of rNDV-GFP was described in the published literature [17]. Sendai FITC-conjugated sheep anti-mouse IgG (F-3008, Sigma) or Alexa virus was grown in eggs and titrated by a hemagglutination assay. FluorÒ 568 goat-rabbit IgG (Invitrogen Molecular Probes). The cells were stained with 1 lg/ml DAPI in PBS for 1 min, washed with PBS, 2.2. Plasmid construction and transfection and mounted on a slide glass with FluoroGuard antifade reagent (Bio-Rad). Images were obtained with a confocal laser scanning The cDNA for human RNase L, OAS1 isoforms p42, p48 and p52, microscope (LSM 700, Carl Zeiss). OAS2 isoforms p69 and p71, and OAS3 p100 were amplified from the RNA of A549 cells following IFN-a treatment. OAS1 p46 cDNA was prepared from human HT1080 fibrosarcoma cells. The RT-PCR 3. Results was conducted using the ImProm II RT-PCR system (Promega) with oligo(dT) and the specific primers shown in the Supplementary Ta- 3.1. The scarce endogenous expression of RNase L in human hepatoma ble 1. The PCR products were digested with HindIII–BamHI (for cells RNase L) or NotI-XbaI (for OAS1 isoforms) and were then subcloned into p3ÂFlag-CMV10 (Sigma). Amplified OAS2 and OAS3 cDNAs Before investigating the antiviral activity of RNase L against were subcloned into pHM6 (Roche). The nuclease-defective RNase HCV, we examined the expression of RNase L in the human hepa- L constructs, R667A, H672A and R462Q were generated by PCR with toma Huh7 cell line and its subclone Huh7.5, both of which sup- primers shown in Table 1.
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