Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells.
We describe a versatile system for monitoring the activity of the NS3-4A serine protease of the hepatitis C virus (HCV) in mammalian cells. The system relies on coexpression of the protease and of an artificial substrate containing a reporter domain and an intracellular targeting sequence separated by a NS3-4A-specific cleavage site. We constructed two different substrates suitable for different applications. The first substrate secretory alkaline phosphatase-1 (SEAP-1) harbors the NS3-4A cleavage site inserted between the SEAP and a membrane anchor featuring an endoplasmic reticulum retention sequence. The arrangement of this substrate is such that SEAP is secreted in the extracellular medium depending on the NS3 protease activity. We show that SEAP-1 can be used to evaluate the activity of NS3-4A inhibitors in living cells. In the second substrate (CD8-1), SEAP is replaced by the extracellular domain of the lymphocyte surface antigen CD8 alpha. The arrangement of this substrate is such that the CD8 alpha domain is transported to the cell surface upon NS3-4Ap cleavage and remains associated with the plasma membrane as an integral membrane protein. We show that CD8-1 can be used for selecting cells capable of supporting HCV replication.
Pacini
Laura
L
IRBM P. Angeletti, Via Pontina km 30.600, 00040 Pomezia, Rome, Italy.
Bartholomew
Linda
L
Vitelli
Alessandra
A
Migliaccio
Giovanni
G
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D016827:CD8 Antigens / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000254:growth & development; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D016827:CD8 Antigens; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases