Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

The role of functional amyloid in the human necroptosis cascade and its modulation by herpesviruses (#140)

Nirukshan Shanmugam 1 , Chi Le Lan Pham 1 , Max Baker 1 , James Brown 2 , Emma Sierecki 2 , Yann Gambin 2 , Margie Sunde 1
  1. University of Sydney, Camperdown, NSW, Australia
  2. EMBL Australia Node in Single Molecule Sciences, School of Medical Science, University of New South Wales, Kensington, NSW, Australia

Functional amyloid complexes are involved in signalling for necroptosis, an inflammatory form of programmed cell death1. The key adapter proteins needed for necroptosis activation are receptor interacting protein kinase 1 (RIPK1), TIR-domain-containing adaptor-inducing interferon-B (TRIF) and the Z-DNA binding protein 1 (ZBP1). These are activated in response to inflammation and microbial infection and subsequently interact with receptor interacting protein kinase 3 (RIPK3), through amino acid sequences called RIP homotypic interaction motifs (RHIMs), to form hetero-oligomeric amyloid fibrils2. This results in activation of RIPK3, phosphorylation of the pseudokinase MLKL and lytic cell death.

 

Viruses such as murine cytomegalovirus (MCMV) and herpes simplex virus 1 (HSV-1) encode RHIM-containing proteins, M45 and ICP6 respectively, that are involved in inhibition of necroptosis in host cells, to ensure viral survival3. The molecular mechanisms underpinning the viral modulation of host necroptosis-associated proteins are the focus of this study.

 

We have produced multiple RHIM-containing fusion proteins with fluorescent and non‑fluorescent tags to study the interaction between host and viral proteins and to distinguish between the formation of homo-oligomeric and hetero-oligomeric amyloid fibrils. Amyloid assembly by the RHIM-containing proteins has been studied by Thioflavin T fluorescence assays, SDS agarose gel electrophoresis, and single molecule confocal spectroscopy. The effect of mutations within the RHIMs of these proteins on self-assembly, and on interactions with other RHIM-containing proteins, have been determined. The large fibrillar complexes formed by the viral and host proteins have been imaged by fluorescence and electron microscopy to gain insight into the mechanism of viral modulation of necroptosis.

 

We have found that the RHIM of the viral protein M45 from MCMV is amyloidogenic and capable of forming heteromeric amyloid structures with host RHIM-containing proteins4. The ICP6 protein from HSV-1 appears to form similar hybrid amyloid-based complexes with host proteins, suggesting a common RHIM-targeting mechanism for viral evasion of the host response.

 

  1. (1) Li et al., 150, 339-50 (2012).
  2. (2) Mompeán et al. Cell. 173, 1244-1253 (2018).
  3. (3) Mocarski et al., Virology. 479-480, 160-6 (2015)
  4. (4) Pham et al., EMBO Rep. e46518. (2019)