A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry

Article

Abstract

Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin–moesin–radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.

Keywords

Anti-viral peptide Ezrin HCV J6/JFH-1 virus Spleen tyrosine kinase (SYK) Moesin Radixin 

Supplementary material

10989_2013_9390_MOESM1_ESM.docx (265 kb)
Supplementary Fig. 1Hydrophobicity analysis of Peptide 1, Hep1 and Peptide 6. Using the computational resource for drug design software, the hydrophobicity of Peptide 1 and Peptide 6 were determined for comparison to the anti-HCV peptide Hep1 allowing the selection of Peptide 1 over Peptide 6 for further analysis. Supplementary material 1 (DOCX 264 kb)
10989_2013_9390_MOESM2_ESM.docx (58 kb)
Supplementary Fig. 2Cellular toxicity assay for anti-HCV peptide 1. Cellular toxicity of anti-HCV Peptide 1 in Huh7.5 cells was analyzed 24 h after peptide treatment using the LDH-cytotoxicity assay (Abcam cat. # ab65393) according to the manufacturers’ specifications. Staurosporine (20nM) treatment or complete lysis of Huh7.5 cells was used as internal positive control as indicated. Toxicity assay data are expressed as mean + SEM, and p < 0.05 was considered statistically significant by the Mann–Whitney test for 3 independent repeat experiments. Supplementary material 2 (DOCX 58 kb)
10989_2013_9390_MOESM3_ESM.docx (155 kb)
Supplementary Fig. 3Anti-HCV Peptide 1 is not a cell penetrating peptide. Huh7.5 cells on cover slips were treated with carboxyfluorescein (FAM)-conjugated-Peptide 1 or Buforin IIb. 24 h after peptide treatment cells were washed with cold PBS and fixed in 2 % formal-saline solution for 10 min at room temperature. After fixation cells were washed twice with PBS, mounted to slides with ProLong® Gold Antifade Reagent with DAPI (Invitrogen cat. # P-36931). Images were then acquired using an Olympus BX51 fluorescence microscope and the Nixon NIS-Element BR3.10 software. A representative image is shown from 3 independently repeat experiments. Supplementary material 3 (DOCX 154 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Terence N. Bukong
    • 1
  • Karen Kodys
    • 1
  • Gyongyi Szabo
    • 1
  1. 1.Department of MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA

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