Development of a cell-based assay for monitoring hepatitis C virus ns3/4a protease activity.
Hepatitis C virus (HCV) NS3/4A (non-structural 3 and 4 B) protease plays a key role in the processing of polyprotein precursor and it becomes an attractive target for antiviral drug discovery. We developed a cell-based assay for monitoring of the NS3/4A protease activity in mammalian cells that is an important step in screening of specific drugs against the protease. The recombinant caspase 3 (rCasp3) was used as the specific substrate for NS3/4A protease. The endogenous cleavage sites in the procaspase 3 molecule were substituted by decapeptides specific for NS3/4A protease. The activation of rCasp3 depended on its specific cleavage by NS3/4A protease and resulted in an apoptosis of stable cells expressing the protease. The difference in cell viability between the cells expressing NS3/4A protease transfected with rCasp3 and the counterparts pretreated with NS3/4A protease inhibitors could be estimated by a spectrophotometry based on 3-(4,5-dimethylthioazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining of cells in microplates. Thus, we developed a simple and cost-effective colorimetric assay for evaluating NS3/4A protease activity enabling the screening of candidate NS3/4A protease inhibitors.
Lei
Y F
YF
Department of Microbiology, Fourth Medical Military University, and Department of Blood Transfusion, Xijing Hospital, Shaanzi, PR China.
Yin
W
W
Yang
J
J
Lv
X
X
Wei
S H
SH
An
Q X
QX
Hu
X B
XB
Xu
Z K
ZK
D000998:Antiviral Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D003124:Colorimetry; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008826:Microbial Sensitivity Tests; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D002352:Carrier Proteins; D053148:Caspase 3; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; 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