Proximity Labelling Identifies Proteins Associated with HSV-2 pUL21 at Early and Late Times After Infection

pUL21 is a conserved and multifunctional alphaherpesvirus tegument protein that is critical for both early and late stages in the herpes simplex virus type 2 (HSV-2) replication cycle; however, how pUL21 participates in these activities is poorly understood. To help elucidate the role of pUL21 in these various activities, we used a proximity-dependent biotin identification (BioID) approach by constructing an HSV-2 strain encoding pUL21 fused to the non-specific biotin ligase, miniTurbo (pUL21mT). Cells infected with this strain were treated with exogenous biotin at early and late times post-infection and biotinylated proteins were affinity-purified and identified by mass spectrometry. This approach enabled the identification of many viral and cellular proteins in proximity to pUL21mT at late times after infection, including those involved in cell adhesion and junction organization, as well as components of the spliceosome and the nuclear envelope. We also utilized this system to identify proteins in proximity to tegument-delivered pUL21mT immediately following viral entry, thereby providing a comprehensive profile of pUL21 proximal interactors at both early and late stages of infection. These proximal interactions were further validated by pUL21 affinity purification experiments, which confirmed that many viral and cellular proteins identified by BioID associate with pUL21. These findings provide insights into the role of HSV-2 pUL21 during infection and highlight BioID as a powerful tool for investigating virus-host interactions at multiple stages of infection.