Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals.
The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANluc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NS5A/B. We proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANluc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-alpha. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals.
Wang
Licui
L
Beijing Institute of Transfusion Medicine, China.
Fu
Qiuxia
Q
Dong
Yafeng
Y
Zhou
Yong
Y
Jia
Shuaizheng
S
Du
Juan
J
Zhao
Fang
F
Wang
Yingli
Y
Wang
Xiaohui
X
Peng
Jianchun
J
Yang
Shuhua
S
Zhan
Linsheng
L
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D049409:Luciferases, Firefly; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus