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Peptides v.88, 2017년, pp.87 - 96   SCI SCIE
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An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides

Peskova, Marie (Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czechia ); Heger, Zbynek (Department of Chemistry and Biochemistry, Mendel University, 613 00 Brno, Czechia ); Janda, Petr (Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czechia ); Adam, Vojtech (Department of Chemistry and Biochemistry, Mendel University, 613 00 Brno, Czechia ); Pekarik, Vladimir (Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno, Czechia );
  • 초록  

    Abstract Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles. Highlights Virolytic assay for pore forming peptides based on Ebola virus-like particles. Applicability to search for Ebola and Marburg virus inactivating compounds. Systematic comparison of pore forming peptides in hemolytic and virolytic assays. Cecropin A/Melittin hybrid (CAM-W) destroys infectivity of lentiviruses. CAM-W effectively lyses Ebola virus-like particles at micromolar concentrations. Graphical abstract The membrane impermeable luciferase substrate furimazine is outside of the virus-like particles while the reporter enzyme is enclosed inside the viral membrane. While the membrane is intact, the substrate remains separated from the luciferase. After the membrane integrity is compromised by the antimicrobial peptide, a pore is formed that allows an ingress of the substrate to the inside of virus like particle. The substrate gets in contact with the enzyme and furimazine chemical conversion is accompanied by the light emission that can be easily detected. [DISPLAY OMISSION]


  • 주제어

    Virus-like particles .   Cytotoxic peptides .   Antimicrobial peptides .   Nanoluciferase .   Hemolytic activity .   Virolytic activity .   Lentivirus .   Ebola .   Marburg.  

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