Severe acute respiratory syndrome-associated coronavirus type 1 (SARS-CoV-1) 3a protein was shown to interact with receptor interacting protein kinase 3 (RIPK3) by immunoprecipitation analysis upon co-expression of viral 3a and RIPK3 in human embryonic kidney 293 (HEK293) cells (Yue Y et al. 2018). Mapping of the interaction between RIPK3 and 3a showed that the kinase domain of RIPK3 (1–326) interacted with SARS-CoV-1 3a, but that the RIP homotypic interaction motif (RHIM) containing C-terminus (327–518) interacted very weakly. Time-lapse confocal microscopy using Cherry-tagged RIP3 in HeLa cells expressing SARS-CoV-1 3a-GFP showed that expression of RIPK3 drives cell death in the presence of SARS 3a. Further, RIPK3-induced oligomerization of SARS-CoV-1 3a (studied with the oligomerization-deficient viral 3a-flag C133A mutant) helped drive necrotic cell death in RIPK3-expressing HEK293, HeLa and 5-Aza-2′-deoxycytidine (5-AD)-treated human alveolar epithelial A549 cells (Yue Y et al. 2018). The A549 cell line is resistant to the traditional necroptotic stimuli, but treatment with hypomethylating agents such as 5-AD induced RIPK3 expression (Yue Y et al. 2018). The results of the study suggest that SARS-Cov-1 3a does not induce cell death in the absence of RIPK3, but induces significant oligomerization-dependent death in the presence of endogenous RIPK3. (Yue Y et al. 2018).
During tumor necrosis factor (TNF)-induced necroptosis, RIPK3 and RIPK1 associate with each other through their RHIM domains into heteromeric RIPK1:RIPK3 complexes that further polymerize into filamentous β-amyloid structures promoting the activation of RIPK3 kinase (Cho Y et al. 2009; Li J et al. 2012). Functionally active RIPK3 activates mixed-lineage kinase domain-like pseudokinase (MLKL), the membrane-disrupting effector of programmed necrosis (Sun L et al. 2012; Murphy JM et al. 2013; Wang H et al. 2014). Other RHIM-containing proteins, such as the TLR3/TLR4 adaptor TRIF (also known as TICAM1) and the DNA sensor DAI/ZBP can form the necroptotic signaling platforms to support activation of RIPK3 and its interaction with MLKL (Kaiser W et al. 2013; Lin J et al. 2016).
This Reactome event shows a scaffolding role of RIPK3 bound to RIPK1 in supporting the formation of SARS-CoV-1 3a oligomers.