RIP1 is a central signaling protein in regulation of TNF-α/TRAIL mediated apoptosis and necroptosis during Newcastle disease virus infection

Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in tumor cells and exerts anti-tumor cytotoxic activity by promoting cell death.In this study, we focus on characterization of the underlying mechanisms of NDVinduced cell death in HeLa cells.We find that NDV Herts/33 strain triggers both extrinsic and intrinsic apoptosis at late infection times.The activation of NF-кB pathway and subsequent up-regulation of TNF-α/TRAIL initiates extrinsic apoptosis, leading to activation of caspase 8 and cleavage of Bid into tBid.tBid transmits the extrinsic apoptotic signals to mitochondria and mediates intrinsic apoptosis, which is hallmarked by cleavage of caspase 9.Moreover, RIP1 is cleaved into RIP1-N and RIP1-C at D324 by caspase 8, and this cleavage promotes apoptosis.Surprisingly, over expression of RIP1 reduces apoptosis and depletion of RIP1 promotes apoptosis, suggesting full length RIP1 is anti-apoptotic.Moreover, necroptosis hallmark protein MLKL is activated by phosphorylation at 12-24 h.p.i., and RIP1 regulates the level of phosphor-MLKL.Immunostaining shows that RIP1 aggregates to stress granules (SGs) at 8-24 h.p.i., and phosphor-MLKL is also recruited to SGs, instead of migrating to plasma membrane to exert its necrotic function.Immunoprecipitation study demonstrates that RIP1 bind to phosphor-MLKL, and depletion of RIP1 reduces the aggregation of MLKL to SGs, suggesting that RIP1 recruits MLKL to SGs.Altogether, NDV infection initiates extrinsic apoptosis via activation of NF-кB and secretion of TNF-α/TRAIL.Activation of caspase 8 by TNF-α/TRAIL and subsequent cleavage of Bid and RIP1 transmit the death signals to mitochondria.Meanwhile, virus subverts the host defensive necroptosis via recruiting phosphor-MLKL by RIP1 to SGs.Thus, RIP1 is a central signaling protein in regulation of apoptosis and necroptosis during NDV infection.