Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity
NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this st...
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th-mahidol.318242018-10-19T11:59:40Z Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity Aninthita Phalaphol Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Ornnuthchar Poungpair Kunan Bangphoomi Nitat Sookrung Potjanee Srimanote Wanpen Chaicumpa Mahidol University Kasetsart University Thammasat University Immunology and Microbiology NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this study, recombinant C-terminal NS3 protein of HCV genotype 3a with endowed helicase activity was produced and used as antigen by selecting VH/VHH display phage clones from an established humanized-camel single domain antibody library that bound specifically to HCV helicase. The VH/VHH derived from phage transfected Escherichia coli clones were linked molecularly to a cell penetrating peptide, i.e., penetratin (PEN). The cell penetrable VH/VHH (transbodies) could reduce the amounts of the HCV RNA released into the cell culture fluid and inside Huh7 cells infected with pJFH1 replicon with a greater effect on the former compared to the latter. Regions and residues of the helicase bound by the transbodies were determined by phage mimotope searching and multiple alignments as well as homology modeling and molecular docking. The epitope of one transbody (PEN-VHH9) encompassed residues 588RLKPTLHGPTPLLYRLGA605 of the domain 3 necessary for helicase activity while another transbody (PEN-VH59) interacted with the areas covering the phenylalanine loop and arginine clamp of the domain 2 which are important for the proper folding of the enzyme as well as nucleic acid substrate binding. Although the molecular mechanisms of the prototypic transbodies on NS3 helicase need further investigation, these transbodies have high potential as novel, safe and mutation tolerable anti-HCV agents. © 2013 . 2018-10-19T04:59:40Z 2018-10-19T04:59:40Z 2013-12-01 Article Journal of Virological Methods. Vol.194, No.1-2 (2013), 289-299 10.1016/j.jviromet.2013.08.032 18790984 01660934 2-s2.0-84884911363 https://repository.li.mahidol.ac.th/handle/123456789/31824 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84884911363&origin=inward |
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Immunology and Microbiology Aninthita Phalaphol Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Ornnuthchar Poungpair Kunan Bangphoomi Nitat Sookrung Potjanee Srimanote Wanpen Chaicumpa Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
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NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this study, recombinant C-terminal NS3 protein of HCV genotype 3a with endowed helicase activity was produced and used as antigen by selecting VH/VHH display phage clones from an established humanized-camel single domain antibody library that bound specifically to HCV helicase. The VH/VHH derived from phage transfected Escherichia coli clones were linked molecularly to a cell penetrating peptide, i.e., penetratin (PEN). The cell penetrable VH/VHH (transbodies) could reduce the amounts of the HCV RNA released into the cell culture fluid and inside Huh7 cells infected with pJFH1 replicon with a greater effect on the former compared to the latter. Regions and residues of the helicase bound by the transbodies were determined by phage mimotope searching and multiple alignments as well as homology modeling and molecular docking. The epitope of one transbody (PEN-VHH9) encompassed residues 588RLKPTLHGPTPLLYRLGA605 of the domain 3 necessary for helicase activity while another transbody (PEN-VH59) interacted with the areas covering the phenylalanine loop and arginine clamp of the domain 2 which are important for the proper folding of the enzyme as well as nucleic acid substrate binding. Although the molecular mechanisms of the prototypic transbodies on NS3 helicase need further investigation, these transbodies have high potential as novel, safe and mutation tolerable anti-HCV agents. © 2013 . |
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Mahidol University |
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Mahidol University Aninthita Phalaphol Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Ornnuthchar Poungpair Kunan Bangphoomi Nitat Sookrung Potjanee Srimanote Wanpen Chaicumpa |
format |
Article |
author |
Aninthita Phalaphol Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Ornnuthchar Poungpair Kunan Bangphoomi Nitat Sookrung Potjanee Srimanote Wanpen Chaicumpa |
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Aninthita Phalaphol |
title |
Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
title_short |
Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
title_full |
Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
title_fullStr |
Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
title_full_unstemmed |
Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity |
title_sort |
humanized-vh/vhh that inhibit hcv replication by interfering with the virus helicase activity |
publishDate |
2018 |
url |
https://repository.li.mahidol.ac.th/handle/123456789/31824 |
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1763492522639753216 |