Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy

Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2011-10, Vol.55 (2), p.127-136
Main Authors: Whitt, Michael A., Mire, Chad E.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biarsenical Lumio labeling
Cell Culture Techniques
Cells, Cultured
Cricetinae
Endocytosis
Fluoresceins - chemistry
Fluorescent Dyes - chemistry
Matrix protein
Microscopy, Confocal - methods
Molecular Sequence Data
Oligopeptides - metabolism
Organometallic Compounds - chemistry
Recombinant Fusion Proteins
Single-Cell Analysis - methods
Staining and Labeling - methods
Tetracysteine tag
Vesiculovirus - chemistry
Vesiculovirus - metabolism
Vesiculovirus - physiology
Viral Structural Proteins - chemistry
Viral Structural Proteins - metabolism
Virus entry
Virus Internalization
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Summary:Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2011.09.002