Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Unravelling Herpesvirus DNA Annealing: Cryo-EM Structure of BALF2 (#340)

Jordan Nicholls 1 2 , Jodi Brewster 2 , Gökhan Tolun 1 2
  1. Centre for Cryo-electron Microscopy of Membrane Proteins, Wollongong, NSW, Australia
  2. School of Chemistry and Molecular Bioscience, and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia

Epstein-Barr Virus (EBV) is a widespread dsDNA γ-herpesvirus, with a 95% global lifetime incidence [1]. EBV infection is life-long and is characterised by latent asymptomatic infection, accompanied by recurrent symptomatic episodes. These episodes involve rapid thousand-fold amplification of the EBV genome, called lytic replication, and are linked to the development of several cancers [1]. Widespread Homologous Recombination (HR) events are an essential process for the formation of lytic replication intermediates and are necessary for correct packaging of mature viral DNA into complete virions [2-4]. HR is also a powerful driver of genetic diversity [5]. Studies focusing on the essential viral mechanisms involved are of great interest as potential antiviral targets.

BALF2 is the essential herpesvirus recombinase responsible for these HR events [6,7]. Specifically, it carries out Single-Strand Annealing (SSA) as the annealase of an Exonuclease-Annealase Two-Component Recombinase (EATR) System [7]. This ATP-independent process is ubiquitously conserved [7]. In EBV, the BGLF5 exonuclease is thought to first expose ssDNA which is then coated by the BALF2 annealase which facilitates annealing to an independently generated, and similarly processed, strand [4, 7, 8]. This is thought to occur during the generation of replication intermediates, and outside of replication in the repair of spontaneous double-stranded DNA breaks [2-4].

BALF2 is a promising candidate for further study as an antiviral target given its essential nature. Unfortunately, very little is understood about the annealing process. Therefore, we aimed to determine the structure of BALF2 in complex with ssDNA as an annealing intermediate to resolve its mechanism of action. In this study, BALF2 was cloned, expressed, and purified. We optimised the formation of a helical nucleoprotein SSA intermediate and solved its structure to 2.2 Å by Cryo-EM. This allowed for the characterisation of the helical architecture, an OB-fold, a zinc binding site, and an active annealing site. It also suggests mechanisms for annealing and cooperative binding. This structure will strongly inform future studies on herpesvirus annealases and the process of SSA, both structural and biochemical, and has great potential as a starting point for structure-based drug design.

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