EBV BALF2 DNA annealing intermediate structure reveals the mechanism of annealing during recombination

Epstein-Barr virus is an oncogenic herpesvirus present in 95% of the global population. It encodes the highly conserved BALF2 protein as an essential member of its replisome. BALF2 is a multifunctional protein which acts as a general single-stranded DNA-binding protein during replication, and as an ATP-independent recombinase involved in the single-strand annealing homologous recombination pathway. Several lines of evidence suggest that homologous recombination is an integral feature of herpesvirus DNA replication, required for the generation of concatemeric replication intermediates, genomic maintenance, and as a major driver of genetic diversity. BALF2 and its homologues are therefore promising antiviral targets. Despite over half a century of research into the herpesvirus annealase proteins, a significant roadblock persists in our understanding of their binding and annealing mechanisms. Here, we present a structure of a BALF2 DNA annealing intermediate, determined to 2.2 Å resolution by cryogenic electron-microscopy (cryo-EM). This structure allowed for the identification and characterisation of an oligonucleotide-binding fold, a zinc-binding loop, an active site of ssDNA-annealing, and suggests a model for cooperative binding and oligomerisation. We also investigated BALF2 through biochemical assays, bioinformatic sequence analysis and molecular dynamics simulations to further characterise regions of the protein’s structure. These findings will strongly inform future studies on herpesvirus annealases and have great potential as a starting point for structure-based drug design.