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Serine Protease Inhibitor AEBSF Reduces Dengue Virus Infection Via Decreased Cholesterol Synthesis T

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Serine Protease Inhibitor AEBSF Reduces Dengue Virus Infection Via Decreased Cholesterol Synthesis T Virus Research 271 (2019) 197672 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis T Liji Sreelathaa,b, Shilu Malakara,c, Pucharee Songprakhona, Atthapan Morchanga, Chatchawan Srisawatb, Sansanee Noisakrand, Pa-thai Yenchitosomanusa, ⁎ Thawornchai Limjindaporna,e, a Siriraj Center of Research Excellence for Molecular Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand b Graduate Program in Medical Biochemistry and Molecular Biology, Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand c Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand d Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand e Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand ARTICLE INFO ABSTRACT Keywords: Dengue virus (DENV) infection has evolved into a major global health menace and economic burden due to its Serine protease inhibitor intensity and geographic distribution. DENV infection in humans can cause a wide range of symptoms including AEBSF dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An antiviral agent that Dengue virus infection is effective against all four serotypes of DENV is urgently needed to prevent and to manage this condition. Cholesterol synthesis Reducing the viral load during the early phase of infection may minimize the chance of patients progressing to more severe DHF or DSS. In this study, we set forth to investigate the anti-viral effect of five commercially available protease inhibitors on DENV infection since both viral and host proteases can contribute to effective viral replication. Previously, the serine protease inhibitor AEBSF [4-(2-aminoethyl) benzene sulfonyl fluoride] has been shown to inhibit DENV NS3 protease activity. The results of the present study revealed that DENV genome replication and protein synthesis were significantly inhibited by AEBSF in a dose-dependent manner. AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl-glutaryl-CoA synthase (HMGCS), 3-hy- droxy-3-methyl-glutaryl-CoA reductase (HMGCR), and low-density lipoprotein receptor (LDLR). Moreover, AEBSF significantly inhibited HMGCR activity and intracellular cholesterol synthesis after DENV infection. The anti-DENV effect of AEBSF was confirmed in all four DENV serotypes and in three different cell lines. These results indicate that AEBSF reduces DENV infection via both viral and host protease activities. 1. Introduction Despite tremendous efforts to develop anti-DENV therapy, none have been approved for clinical use. An antiviral agent against DENV DENV is a flavivirus that belongs to the Flaviviridae family, and that should achieve comparable viral inhibition against all DENV serotypes. comprises four serologically related, but antigenically distinct ser- Pre-clinical and clinical studies demonstrated the involvement of both otypes. The viral genome is composed of positive-sense RNA. DENV viral and host factors in the severity of this disease. Host protease is infection can cause a broad spectrum of clinical symptoms including shown to play important roles in virus entry, uncoating, viral protein dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock production, maturation, and disease induction (Bartenschlager and syndrome (DSS) (Kautner et al., 1997). Unplanned urbanization and Miller, 2008) while viral protease facilitates viral genome replication global travel contribute to the global distribution of Aedes mosquito and polyprotein processing (Murthy et al., 1999). Interventions that can vectors across the world (Guzmán and Kouri, 2002). It is estimated that inhibit or obstruct the aforementioned functions of these proteases may DENV infection affects approximately 100 million people annually in be an effective approach to prevent or to treat DENV infection. Anti- over 120 countries (Bhatt et al., 2013). DENV therapy research suggests that antiviral agents can inhibit viral ⁎ Corresponding author at: Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok 10700, Thailand. E-mail address: [email protected] (T. Limjindaporn). https://doi.org/10.1016/j.virusres.2019.197672 Received 7 December 2018; Received in revised form 10 April 2019; Accepted 17 July 2019 Available online 03 August 2019 0168-1702/ © 2019 Elsevier B.V. All rights reserved. L. Sreelatha, et al. Virus Research 271 (2019) 197672 proteins including viral capsid, viral protease, viral helicase, viral me- glutamine, 1% Non-Essential Amino Acid (NEAA), 1 mM sodium pyr- thyltransferase, viral polymerase, and host targets (Lim et al., 2013). Of uvate, and antibiotics. the antiviral agents that have been investigated, protease inhibitors showed promise as an effective treatment approach. The antiviral ac- 2.3. Cell viability assay tivity of protease inhibitors has been documented in human im- munodeficiency virus (HIV) and hepatitis C virus (HCV) by targeting To evaluate inhibitor cytotoxicity, cells were treated with serially viral enzymes (Agbowuro et al., 2017; Menéndez-Arias, 2010; Patick diluted protease inhibitors in a 96-well plate for 24 h at 37 °C with 5% and Potts, 1998; Soriano et al., 2008). Most of the protease inhibitors CO2. To assess cell viability, cells were inoculated with DENV at a developed against DENV are targeted at viral enzymes, particularly multiplicity of infection (MOI) of 1 for 2 h. After incubation, cells were viral NS2B/NS3 protease (Wu et al., 2015; Yang et al., 2011). washed twice with stock medium and treated with protease inhibitors In this study, five protease inhibitors representing different classes at different concentrations for 24 h. After incubation, 10 μlof of host proteases were tested for their anti-DENV properties. Serine Prestoblue® Reagent (Invitrogen, Carlsbad, CA, USA) was added to each protease inhibitors [4-(2-aminoethyl) benzene sulfonyl fluoride well. Plates were then incubated for 30 min. at 37 °C after which cell (AEBSF) and aprotinin], a cysteine protease inhibitor [trans-epox- viability was determined by measuring the absorbance at 570/595 nm ysuccinyl-L-leucylamido (4-guanidino) butane (E-64)], a metallopro- in a microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, tease inhibitor [ethylene diamine tetra acetic acid, disodium salt USA). Mock treated cells were used as baseline for the comparison with (EDTA)], and a cysteine-serine protease inhibitor (leupeptin hemi- protease inhibitor-treated cells. To measure cytotoxicity, dose response sulfate) were evaluated in this study. The antiviral activities of these curves were plotted with GraphPad Prism 5 (GraphPad Software Inc., protease inhibitors against DENV were screened by ELISA, western San Diego, CA, USA). blotting and focus forming unit (FFU) assay to determine intracellular DENV E antigen, DENV NS3 protein and mature virus production, re- 2.4. Enzyme-linked immunosorbent assay spectively. Our results demonstrated that AEBSF treatment could in- hibit DENV infection by suppressing both viral RNA and protein Cells were inoculated with DENV2 at an MOI of 1 for 2 h in a 96- synthesis in a dose-dependent manner. Importantly, AEBSF inhibited all well plate. After incubation, cells were washed and treated with pro- four serotypes of DENV regardless of the cell types being infected. tease inhibitors at different concentrations for 24 h. The cells were then AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl- fixed with 3.6% formaldehyde, permeabilized with 0.1% Triton-X 100, glutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methyl-glutaryl-CoA re- and blocked endogenous peroxidase with 3% hydrogen peroxide. Cells ductase (HMGCR), and low-density lipoprotein receptor (LDLR). were incubated with antibody against DENV E protein (4G2) for 1 h Moreover, AEBSF significantly inhibited HMGCR activity and in- followed by incubation with HRP-conjugated secondary antibody for tracellular cholesterol synthesis after DENV infection. Taken together, 30 min. at 37 °C. Substrate 3,3′,5,5′-tetramethylbenzidine (TMB) these findings portend a novel strategy for the development of new anti- (Invitrogen) was added to the cells followed by sulfuric acid (H2SO4)to DENV therapy. stop the reaction. The absorbance, which represents intracellular DENV E, was measured at 450/620 nm in a microtiter plate reader (BioTek 2. Materials and methods Instruments). 2.1. Cell culture and viruses 2.5. Focus forming unit (FFU) assay HepG2 (ATCC® HB-8065™), A549 (ATCC® CCL-185™), and Virus supernatants collected from protease inhibitors-treated and EA.hy926 (ATCC® CRL-2922™) cell lines were purchased from untreated DENV-infected cells were serially diluted and inoculated into American Type Culture Collection (ATCC; Manassas, VA, USA). HepG2 Vero cells for 2 h. Overlay medium 1.5% carboxy methyl cellulose and A549 cell lines were maintained in Dulbecco’s Minimum Essential (Sigma, USA) was added, and the cells were cultured for 72 h at 37 °C. Medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, Cells were fixed with 3.6% formaldehyde and permeabilized with 0.1% USA) supplemented with 10% fetal bovine
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