Oral Presentation 12th Australasian Virology Society Meeting 2024

High-resolution structure of the Circovirus replication associated protein (#36)

Babu Nath 1 , Shubhagata Das 2 , Daniel Luque 3 , Crystall Swarbrick 1 , Shane Raidal 4 , Jade Forwood 5
  1. Gulbali Institute, Charles Sturt University., Research Fellow, Wagga Wagga, NSW, Australia
  2. School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Lecturer, Wagga Wagga, NSW, Australia
  3. Electron Microscope Unit Mark Wainwright Analytical Centre Chemical Sciences Building (F10) & School of Biomedical Sciences, Faculty of Medicine & Health , Associate Director and senior lecturer, Sydney, NSW, Australia
  4. School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Professor in Pathobiology, Wagga Wagga, NSW, Australia
  5. Gulbali Institute, School of Dentistry and medical Sciences, Charles Sturt University., Professor in Biochemistry, Wagga Wagga, NSW, Australia

Circoviruses are ubiquitous and can infect a wide range of mammal and avian hosts. The replication associated protein (Rep) from these viruses is responsible for initiating genome replication using the rolling circle replication (RCR) mechanism. Rep carries out this function through its two functional domains, an endonuclease domain, and a helicase domain, responsible for nicking and ligating ssDNA and unwinding double-stranded DNA (dsDNA). The aim of this study was to recombinantly express Beak and feather disease virus (BFDV) Rep in E. coli and solve the high-resolution structure of Rep through cryo-EM. The structure demonstrates full-length Rep assembles in a hexameric ring similar to that of the viral helicases from the superfamily 3 (SF3) of ATPases Associated with diverse cellular Activities (AAA+). Further 3D classification and refinement has allowed two conformations of the complex to be resolved at 3.4 and 3.7 Å resolution and a model for the full Rep complex. Analysis of the structure has shown the presence of an oligonucleotide loaded on the helicase domain and allowed the identification of three residues implicated in this interaction. The significance of these residues has been tested by the production and purification of point mutants to further probe its capacity for ssDNA binding and replication. This information is important for defining the molecular mechanisms that underlie their functions and would also open new avenues for the design of new antiviral strategies against these viruses.