Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network

Protein-protein interactions (PPIs) are crucial for various biological processes. Epstein-Barr virus (EBV) proteins typically form complexes, regulating the replication and persistence of the viral genome in human cells. However, the role of EBV protein complexes under physiological conditions remains unclear. In this study, we performed comprehensive analyses of EBV PPIs in living cells using the NanoBiT system. We identified 195 PPIs, many of which have not previously been reported. Computational analyses of these PPIs revealed that BLRF2, which is only found in gammaherpesviruses, is a central protein in the structural network of EBV tegument proteins. To characterize the role of BLRF2, we generated two BLRF2 knockout EBV clones using CRISPR/Cas9. BLRF2 knockout significantly decreased the production of infectious virus particles, which was partially restored by exogenous BLRF2 expression. In addition, self-association of BLRF2 protein was found, and mutation of the residues crucial for the self-association affected stability of the protein. Our data imply that BLRF2 is a tegument network hub that plays important roles in progeny virion maturation.