title: Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity.
abstract: Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors.
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Lei
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Haiwei
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Qiao
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Peijun
authors.initials: P
authors.lastname: Han
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.
authors.forename: Zhikai
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an, China.|Department of Blood Transfusion, Xijng Hospital, Fourth Military Medical University, Xi'an, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Yin
mesh_terms: D000818:Animals; D003199:Computer Systems; D004195:Disease Models, Animal; D016174:Hepacivirus / Q000201:enzymology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000821:virology; D008156:Luciferases / Q000378:metabolism; D008822:Mice, Transgenic; D009842:Oligopeptides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D008156:Luciferases; D009842:Oligopeptides; D011392:Proline; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide