The crystal structure of the herpes virus ICP8 protein in complex with single-stranded DNA reveals the molecular...

The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8Δ60) bound to poly(dT)₂₅ or poly(dA)₂₅ ssDNA at 3.0 to 3.1 Å resolution, along with higher-resolution apo structures of surface-entropy-reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, π-stacking and phosphate backbone interactions. In the poly(dT)₂₅ complex, coordination of a Zn²⁺ ion stabilizes the zinc finger motif; whereas, in the poly(dA)₂₅ complex, Zn²⁺ displacement promotes disulfide bond formation that effectively locks the protein into an altered conformation. Microscale thermophoresis and label-free differential scanning fluorimetry reveal a strong preference for pyrimidine-rich sequences, with nanomolar affinity for poly(dT)₂₅ and micromolar for poly(dA)₂₅. Structural modeling identified Y543, R576, R772, R793, Y988, and F998 as key DNA-contact residues. Alanine substitutions caused severe replication defects, particularly for R772A, Y988A, and F998A. ssDNA binding induces ∼26 Å displacement and ∼35 degree rotation of the C-terminal domain and ordering of flexible loops, suggesting a mechanism for cooperative filament assembly. These structures define the molecular determinants of ICP8-ssDNA recognition, reveal thymidine bias and provide a framework for targeting ICP8-mediated functions in herpesvirus replication.