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Probing herpesvirus membrane glycoprotein interactions using proximity biotinylation (993.2)
Author(s) -
Connolly Sarah,
Lajko Michelle,
Haddad Alexander
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.993.2
Subject(s) - biotinylation , biotin , vesicle associated membrane protein 8 , viral entry , glycoprotein , biology , membrane protein , microbiology and biotechnology , fusion protein , lipid bilayer fusion , cell membrane , herpesvirus glycoprotein b , viral membrane , biochemistry , chemistry , cell , virus , viral envelope , viral replication , membrane , virology , recombinant dna , gene
Herpesviruses cause significant disease within the human population, ranging from simple cold sores to life‐threatening lymphomas. The critical first step of any viral infection is entry into a host cell. For herpesviruses, the conserved mechanism of entry is initiated when glycoproteins embedded in the viral membrane bind to specific cellular membrane receptors. This receptor binding elicits interactions among viral membrane glycoproteins that trigger the viral fusion protein to execute virus‐cell membrane fusion, by inserting into the cell membrane and refolding from a prefusion to a postfusion conformation. Details of the viral protein interaction cascade are poorly understood because the interactions are likely transient and/or low affinity. To examine how the proteins interact, we adapted a proximity biotinylation assay (ProB). In this assay, one protein is linked to a bacterial biotin ligase and a second candidate protein is linked to a target acceptor peptide. If the proteins interact during membrane fusion, the ligase on the first protein catalyzes site‐specific biotinylation of the acceptor peptide on the second candidate. Labeling is covalent and occurs within live cells, thus weak and transient interactions can be detected. Positive controls show strong biotinylation, indicating that proximity of the viral membrane proteins can be detected. Unexpectedly, proteins that do not functionally interact also show a lower level of biotinylation. The results demonstrate the special circumstances that must be considered when examining interactions among proteins that are constrained within the two‐dimensional boundaries of a membrane.