Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity
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Morgan M. Martin
and François Jean
Abstract
The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic α-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.
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©2006 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- Caspase-containing complexes in the regulation of cell death and inflammation
- Regulation of human cathepsin B by alternative mRNA splicing: homeostasis, fatal errors and cell death
- The peptidases from fungi and viruses
- C. elegans as a model system to study the function of the COG complex in animal development
- Functional responses of bone cells to thrombin
- Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties
- Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite
- Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata
- Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity
- Treatment of MCF-7 cells with taxol and etoposide induces distinct alterations in the expression of apoptosis-related genes BCL2, BCL2L12, BAX, CASPASE-9 and FAS
- Proteolytic mechanism of a novel mitochondrial and chloroplastic PreP peptidasome
- Tripeptidyl-peptidase I in health and disease
- Molecular and functional analysis of new members of the wheat PR4 gene family
- C-Terminal truncations of syncytin-1 (ERVWE1 envelope) that increase its fusogenicity
- Disease processes may be reflected by correlations among tissue kallikrein proteases but not with proteolytic factors uPA and PAI-1 in primary ovarian carcinoma
- Heparin modulation of human plasma kallikrein on different substrates and inhibitors
- Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn
- Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice