Necroptosis, an inflammatory form of programmed cell death, is responsible for the regulation of viral infections and occurs in response to cell or tissue damage. Dysregulation of necroptosis is also associated with several disease states, including inflammatory bowel disease and cancer. The formation of functional amyloid fibrils, containing Receptor Interacting Protein Kinase 3 (RIPK3) and other adapter proteins, is crucial to the signalling pathways that lead to necroptosis. These functional amyloid fibrils form as a result of protein:protein interactions between Receptor interacting protein Homotypic Interaction Motifs (RHIMs) found within RIPK3 and the necroptosis adapter proteins. Z-DNA binding protein 1 (ZBP1), is the adapter protein crucial to the necroptotic response to viral infection. Herpesviruses can manipulate the necroptosis pathway through the expression of viral RHIM‑containing proteins. Herpes Simplex Virus-1 expresses the RHIM-containing protein ICP6. ICP6 can inhibit necroptosis in human cells but fails to do so in murine cells, indicating key differences in the human and murine necroptosis pathways and interactions. Investigating the molecular basis for the initiation and inhibition of the human and murine necroptosis pathways could guide the development of therapeutics that manipulate necroptosis in disease states.
We have investigated the ability of ICP6 to form hybrid amyloid fibrils with host RHIM-containing proteins. Protein:protein interactions between ICP6 and the host RHIM‑containing proteins have been characterised using amyloid assembly assays, electron microscopy and confocal coincidence spectroscopy. The formation and stability of homomeric and heteromeric host:viral RHIM‑dependent assemblies have been investigated using semi‑denaturing agarose gel electrophoresis and probing the degree of depolymerisation of the amyloid structure as a function of detergent concentration. Confocal microscopy has confirmed the heteromeric nature of these assemblies. The interactions of human and murine RIPK1, RIPK3 and ZBP1 with ICP6 have been compared and differences in assembly reveal the likely basis for the species-specific responses to HSV-1 infection. ZBP1 is identified as the key adapter protein for ICP6 interactions and circular dichroism and electron microscopy studies have been utilised to probe the conformational changes of ZBP1 in a monomeric and fibrillar state.