Two distinct protein-protein interfaces drive cooperative binding of the herpes simplex virus protein ICP8 to ssDNA, filament formation and annealing essential for viral replication.

Herpes simplex virus (HSV) replicates by forming DNA concatemers using a single strand annealing mechanism mediated by the multifunctional HSV protein ICP8. ICP8 binds cooperatively to form nucleoprotein filaments on ssDNA and promotes annealing of complementary single strands. These functions are believed to be mediated by ICP8:ICP8 protein-protein interactions (PPIs) between the flexible C-terminal domain of ICP8 and the N-terminal domain of a second ICP8 monomer. We previously presented genetic evidence for an ICP8:ICP8 PPI hotspot between two hydrophobic regions and showed that it is essential for the cooperative binding of ICP8 to ssDNA, ICP8 filament formation in vitro, and viral replication. This involves interactions between F1142, N1143, and F1144 (FNF) in the C terminal disordered tail of ICP8 and the hydrophobic residues (F843 and W844) in the head region of the N-terminal domain on a second molecule of ICP8. In this article, we propose an additional second distinct PPI hotspot involving residues R922, within the body region of the N-terminal domain of ICP8, and D1087, within the C-terminal domain (CTD). Biophysical studies using thermal melting assays and microscale thermophoresis support an interaction between the ICP8-CTD and the N-terminal domain of ICP8 and show that mutations within each of these hotspots disrupt the interactions. Furthermore, we demonstrate that a construct consisting of the ICP8-CTD can antagonize the assembly of ICP8 filaments in vitro, block viral replication compartment formation and virus production, suggesting that these PPI hotspots may be useful in the development of new antiviral therapies.