Soutenance de thèse : Structural and Functional Characterisation of the Vaccinia Virus PLD- fold endonuclease K4, telomere-binding protein I1 and the DNA Polymerase Complex E9A20D4

Par Henri Gröger (IBS/Groupe Machines de Réplication Virale)

Poxviruses, such as the vaccinia virus (VACV) and the monkeypox virus, are large, enveloped dsDNA viruses from the orthopoxvirus genus that replicate entirely within the host cytoplasm. The 2022 and 2024 outbreaks of mpox, caused by clade IIb and Ib, respectively, have revealed the lack of efficient antivirals and underlined the urgency of understanding poxvirus biology. The poxvirus genome is flanked by short, inverted complementary hairpin telomeres that feature mismatched bases and insertions essential for viral replication.

This thesis presents the structural and functional characterisation of three proteins central to poxvirus DNA metabolism : the E9A20D4 DNA polymerase holoenzyme, and two telomere-interacting proteins, the PLD-fold nuclease K4 and I1. The project initially focused on the VACV polymerase holoenzyme, but was reoriented towards the telomere-interacting proteins following the publication of numerous competing mpox polymerase structures.

Having established that VACV polymerase activity requires K⁺ and is inhibited by Na⁺, I undertook a structure determination of the E9A20D4 polymerase holoenzyme bound to template DNA, primer and incoming nucleotide in the presence of K⁺, using single-particle cryogenic electron microscopy (cryo-EM). I obtained both the structure of the complex E9^exo−A20D4 as well as the structure of E9^exo− alone bound to the primer-template DNA. The structures in the presence of K⁺ appear identical to published structures in the presence of Na⁺. However, I identified an ion binding site in the exonuclease domain of E9. The thumb domain is disordered in the DNA-free structure, partially disordered in DNA-bound E9 and ordered in the holoenzyme-DNA complex. SAXS data indicate conformational flexibility, with more open conformations of E9A20D4 lacking an E9-D4 interface, while mass photometry reveals partial dissociation of E9A20D4 at low concentrations, even in the presence of substrate.

Using cryo-EM, I report the first structures of K4 in both apo and DNA-bound states, revealing that the active site is occluded by an orthopoxvirus-specific C-terminal extension of the PLD fold that is displaced upon DNA binding. Biochemical characterisation demonstrates that K4 functions as a DNA-specific endonuclease with a preference for single-stranded DNA and hairpin loops.

I also report the first cryo-EM structure of I1 bound to DNA. I1 is known to bind to viral telomeres and is essential for virion maturation. Cryo-EM data showed the presence of dimers where the head domains 2 and 3 of I1 interact with the DNA duplex through electrostatic interactions, while the N-terminal domain predicted to be α-helical remains disordered. In solution, isolated I1 or I1 bound to DNA forms higher-order assemblies, predominantly tetramers, but also octamers.

Altogether, these findings substantially advance the molecular understanding of poxvirus biology, providing a foundation for future mechanistic studies and the rational development of antiviral strategies against emerging orthopoxvirus infections.