Necroptosis is a form of programmed cell death that functions to commit cells to death during viral infections when apoptosis pathways are inhibited [1]. Necroptosis relies on the formation of a functional amyloid signalling complex comprised of the receptor interacting protein kinase 3 (RIPK3) and other adapter proteins [2, 3]. These structured protein assemblies are stabilised by interactions between RIP Homotypic Interaction Motifs (RHIMs) in the component proteins [3].
The murine cytomegalovirus is known to encode a viral RHIM containing protein, M45, which can interact with RIPK3 and enable viral evasion of necroptosis [4, 5]. This work assesses the importance of residues throughout the RHIM to interactions during viral inhibition of necroptosis by employing tetra alanine mutations scanning across residues flanking the core tetrad in fluorescently-tagged forms of M45 and RIPK3. Homomeric and heteromeric interactions were assessed using Thioflavin T assays in conjunction with fluorescence microscopy and single molecule-confocal spectroscopy, while the morphology and stability of assemblies were interrogated with the use of electron microscopy and sodium dodecyl sulfate agarose gel electrophoresis.
The results of this work not only indicate that residues beyond the core tetrad of RIPK3 are essential to homomeric interaction, in alignment with the recently published structures of the homomeric RIPK3 amyloid [6, 7], but importantly demonstrate the contribution of residues flanking the core tetrad of M45 to heteromeric interactions with RIPK3.
This work has identified the extent of the large interaction interface between RIPK3 and M45 that underpins the structural basis for viral inhibition of necroptosis.