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 language | English (US) |
|---|---|
| Pages (from-to) | 127-136 |
| Number of pages | 10 |
| Journal | Methods |
| Volume | 55 |
| Issue number | 2 |
| DOIs | |
| State | Published - Oct 2011 |
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Keywords
- Biarsenical Lumio labeling
- Endocytosis
- Matrix protein
- Tetracysteine tag
- Virus entry
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy. / Whitt, Michael A.; Mire, Chad.
In: Methods, Vol. 55, No. 2, 10.2011, p. 127-136.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy
AU - Whitt, Michael A.
AU - Mire, Chad
PY - 2011/10
Y1 - 2011/10
N2 - 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.
AB - 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.
KW - Biarsenical Lumio labeling
KW - Endocytosis
KW - Matrix protein
KW - Tetracysteine tag
KW - Virus entry
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UR - http://www.scopus.com/inward/citedby.url?scp=80055097436&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2011.09.002
DO - 10.1016/j.ymeth.2011.09.002
M3 - Article
VL - 55
SP - 127
EP - 136
JO - Methods
JF - Methods
SN - 1046-2023
IS - 2
ER -