Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes

ACS Chem Biol. 2019 Aug 16;14(8):1836-1844. doi: 10.1021/acschembio.9b00493. Epub 2019 Jul 26.


Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoacyltransferases / metabolism
  • Animals
  • Bacterial Proteins / metabolism
  • Cell Line, Tumor
  • Cysteine Endopeptidases / metabolism
  • Dogs
  • Endosomes / metabolism
  • Fluorescent Dyes / chemistry*
  • Influenza A virus / physiology*
  • Madin Darby Canine Kidney Cells
  • Membrane Proteins / metabolism*
  • Mice
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods
  • Peptides / chemistry*
  • Protein Transport
  • Rhodamines / chemistry*
  • Virus Internalization


  • Bacterial Proteins
  • Fluorescent Dyes
  • Membrane Proteins
  • Peptides
  • Rhodamines
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases