Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity.
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 alpha-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.
Martin
Morgan M
MM
Department of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, 3559-2350 Health Sciences Mall, Vancouver, V6T 1Z3, Canada.
Jean
François
F
D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D016174:Hepacivirus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / 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; 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