Examination of herpesvirus entry glycoprotein interactions using proximity biotinylation

Herpesvirus entry into cells begins when viral membrane glycoproteins bind to cellular receptors. This receptor binding elicits a cascade of interactions among conserved viral membrane glycoproteins that triggers virus-cell membrane fusion. Details of the viral protein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation (ProB) is a promising technique for dissecting the glycoprotein interactions required for entry. In ProB, one protein is linked to a bacterial biotin ligase and a second protein is linked to a target 15 amino acid acceptor peptide (AP). If the proteins interact, the ligase on the first protein catalyzes site-specific biotinylation of the AP on the second protein. Evidence of interaction can be detected by western blotting with streptavidin to visualize biotinylated proteins. Labeling is covalent and occurs within live cells, thus weak and transient interactions can be detected. The short length of AP reduces the risk of altering the native conformation of the glycoproteins. Sensitivity and specificity are concerns for all interaction assays. To establish optimal conditions for ProB, the biotin ligase and AP were linked to herpes simplex virus gD and Epstein Barr virus gB. These oligomeric herpesvirus proteins do not interact functionally and assaying for a heterotypic interaction between them serves as a negative control to demonstrate specificity of the ProB assay. Homotypic interactions between their monomers serve as a positive control to demonstrate sensitivity. The positive controls show strong biotinylation, indicating that proximity of viral membrane proteins can be detected. Unexpectedly, the negative controls also showed biotinylation, albeit at a lower level. These 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. The lab is currently investigating ways to limit the heterotypic interaction and designing constructs to further test ProB validity for membranerestricted protein interactions