Abstract
Necroptosis is an alternative programmed cell death pathway that is unleashed in the absence of apoptosis and mediated by signaling complexes containing receptor-interating protein kinase 1 (RIPK1) and RIPK3. This form of cell death has recently been implicated in host defense system to eliminate pathogen-infected cells. However, only a few viral species such as herpes simplex virus (HSV) and cytomegalovirus (CMV) have evolved mechanisms inhibiting necroptosis to overcome host antiviral defense, which is important for successful pathogenesis. Here, we show that the γ-herpesvirus Epstein–Barr virus (EBV) blocks necroptosis in EBV-infected human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells. Our findings indicate that EBV-encoded latent membrane protein 1 (LMP1), which lacks an RIP homotypic interaction motif (RHIM) domain, has mechanisms distinct from RHIM signaling competition to inhibit this necroptotic pathway. Intriguingly, LMP1 interacts directly with both RIPK1 and RIPK3 through its C-terminal activation region. More importantly, LMP1 can modulate the post-translational modification of the two receptor-interacting proteins. We then show that LMP1-mediated promotion of K63-polyubiquitinated RIPK1, suppression of RIPK1 protein expression and inhibition of K63-polyubiquitinated RIPK3 induced a switch in cell fate from necroptotic death to survival. These findings provide direct evidence for the suppression of necroptosis by EBV and define a mechanism of LMP1 to interrupt the initiation process of necroptosis before necrosome formation.
Highlights
Programmed necrosis or necroptosis has emerged as a novel form of programmed cell death that is independent of caspase activity
Our results showed that T/S/Z induction led to massive cell death of NP460hTERT cells and this pattern of cell death was prevented to a certain extent by the necroptosis specific inhibitor Nec-1 or GSK’872 (Fig. 1a, b), indicating that NP460hTERT cells were sensitive to T/S/Z-induced necroptosis
As RIPK3 phosphorylation at S227 and mixed lineage kinase domain-like (MLKL) phosphorylation at T357/S358 is critical for the induction of necroptosis, we performed an immunoblot analysis of phosphorylated RIPK3 (p-RIPK3) and phosphorylated MLKL (p-MLKL) as well as markers of apoptosis including cleaved caspase-3 and cleaved caspase-8 to confirm that the type of cell death is necroptosis rather than apoptosis
Summary
Programmed necrosis or necroptosis has emerged as a novel form of programmed cell death that is independent of caspase activity. The best characterized necroptosis pathway is triggered by tumor necrosis factor α (TNFα), which requires the receptorinterating protein kinase 1 (RIPK1) and RIPK31. In TNFα-induced necroptosis, RIPK1 and RIPK3 form a. Ser[227] is required for human RIPK3 to recruit and phosphorylate downstream substrate protein mixed lineage kinase domain-like (MLKL)[3]. MLKL forms an oligomer that moves from the cytosol to the plasma and intracellular membranes[4], thereby disrupting membrane integrity and resulting in necrotic death. In addition to RIPK1 and RIPK3, the RHIM is found in TRIF5, DAI/ZBP16, and ICP67,8, which can interact with RIPK3 to form RHIM-dependent signaling complexes. Official journal of the Cell Death Differentiation Association
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