Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications.
OBJECTIVE
To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents.


METHODS
We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems.


RESULTS
The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a.


CONCLUSIONS
The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.
Mao
Hong-Xia
HX
Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Lan
Shui-Yun
SY
Hu
Yun-Wen
YW
Xiang
Li
L
Yuan
Zheng-Hong
ZH
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D005838:Genotype; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008113:Liver Neoplasms; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus