# Proximity labelling identifies proteins associated with HSV-2 pUL21 at early and late times after infection

## TL;DR

This study uses a proximity labeling technique to identify proteins interacting with HSV-2 pUL21 during early and late stages of infection, revealing new insights into its functions.

## Contribution

The study introduces a BioID approach to identify pUL21 interactors at different stages of HSV-2 infection, revealing new roles for pUL21.

## Key findings

- pUL21 interacts with proteins involved in cell adhesion, junction organization, and the spliceosome at late stages of infection.
- Tegument-delivered pUL21 interacts with cellular and viral proteins immediately after viral entry.
- BioID confirmed interactions that were further validated by affinity purification experiments.

## Abstract

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.

The herpesvirus tegument is a subvirion compartment located between the capsid and the envelope and, in the case of HSV, contains about 70 distinct proteins; roughly 20 of these are encoded by the virus genome and 50 are encoded by the host cell genome. During virion entry, the tegument contents are delivered to the host cell cytoplasm. Beyond their immediate roles after infection, tegument proteins function throughout infection and support, amongst other things, the transcription of virus genes, evasion of innate and adaptive immune responses, and virion assembly. Despite great progress in understanding tegument protein function, much remains to be learned, particularly at the earliest stages of infection. We reasoned that the identification of cellular and viral proteins in proximity to the HSV-2 tegument protein pUL21 would provide insight into pUL21 functions. Here, we have taken a BioID approach to identifying pUL21 interaction partners at early and late stages of infection. Our findings support previously established functions of pUL21 in capsid nuclear egress and as a phosphatase adapter protein and reveal potential new roles for pUL21 in the modulation of RNA processing and cell-to-cell spread of infection.