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

Michael A. Whitt, Chad E. Mire

Research output: Contribution to journalReview article

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)127-136
Number of pages10
JournalMethods
Volume55
Issue number2
DOIs
StatePublished - Oct 2011

Keywords

  • Biarsenical Lumio labeling
  • Endocytosis
  • Matrix protein
  • Tetracysteine tag
  • Virus entry

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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