Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

RHIM-based functional amyloid assemblies in viral evasion of host cell death (#256)

Max ODG Baker 1 2 , Chi Le Lan Pham 1 , Nirukshan Shanmugam 1 , Thu Vu 1 , Emma Sierecki 3 , Yann Gambin 3 , Megan Steain 2 , Margie Sunde 1
  1. Discipline of Pharmacology, University of Sydney, Camperdown, New South Wales, Australia
  2. Discipline of Infectious Diseases and Immunology, University of Sydney, Camperdown, New South Wales, Australia
  3. Single Molecule Science, University of New South Wales, Kensington, NSW, Australia

The Receptor Homotypic Interaction Motif (RHIM) is a conserved amino acid sequence, found in four human proteins, which mediates the assembly of a heteromeric signalling complex known as the necrosome that is required for certain types of programmed cell death [1]. The necrosome has a fibrillar amyloid structure. This RHIM-mediated amyloid structure is primarily responsible for the induction of cell death by programmed necrosis, termed necroptosis, but recent findings indicate that it may also play a role in the induction of other cell death pathways, including apoptosis and pyroptosis.

Herpesviruses, highly seropositive DNA viruses that impose significant medical and economic burden, require living host cells to function. To this end, they have evolved multiple mechanisms to counteract host cell death pathways [2]. Herpesviruses express proteins that contain RHIM sequences which interact with human RHIM proteins to form heteromeric, interspecies amyloid complexes that abrogate the signalling capabilities of the human proteins [3]. Here we report the characterisation of a novel RHIM-containing protein from Varicella Zoster virus known as ORF20. Using a range of biophysical techniques, including fluorescence assays, electron microscopy and single molecule spectroscopy, we have shown that the ORF20 RHIM drives formation of homomeric amyloid fibrils and that it interacts with human RHIM proteins to form heteromeric complexes in vitro. We have also validated the interaction between ORF20 and these human proteins in cell-based experiments. In parallel, we have demonstrated the ability of this virus to inhibit cell death through the activity of ORF20 protein, but find that unlike other viral RHIM proteins, ORF20 inhibits apoptosis, not necroptosis, and that this activity is not solely dependent on RHIM interactions. The characterisation of this novel RHIM demonstrates the importance of RHIM-based signalling in multiple human cell death pathways, as well as the diversity of viral strategies to overcome the complexity of signalling driving these processes.

  1. Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsaio YS, et al. Cell 159, 339-350 (2012).
  2. Baker MODG, Shanmugam N, Pham CLL, Strange M, Steain M, Sunde M. Semin Cell Dev Biol, https://doi.org/10.1016/j.semcdb.2018.05.004 (2018).
  3. Pham CLL, Shanmugam N, Strange M, O’Carroll A, Brown JP, Sierecki E, et al. EMBO Rep, https://doi.org/10.15252/embr.201846518 (2018).