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J Virol Methods. 2015 Sep 1;221:81-9. doi: 10.1016/j.jviromet.2015.04.031. Epub 2015 May 6.

Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

Author information

1
DePaul University, Department of Biological Sciences, Chicago, IL, USA.
2
DePaul University, Department of Health Sciences, Chicago, IL, USA.
3
DePaul University, Department of Biological Sciences, Chicago, IL, USA; DePaul University, Department of Health Sciences, Chicago, IL, USA. Electronic address: sconnol6@depaul.edu.

Abstract

Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

KEYWORDS:

Herpesvirus; Protein–protein interactions; Proximity biotinylation; Virus entry

PMID:
25958131
PMCID:
PMC4469388
DOI:
10.1016/j.jviromet.2015.04.031
[Indexed for MEDLINE]
Free PMC Article

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