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, 24 (4), 615-625

Induction of Necroptotic Cell Death by Viral Activation of the RIG-I or STING Pathway

Affiliations

Induction of Necroptotic Cell Death by Viral Activation of the RIG-I or STING Pathway

Suruchi N Schock et al. Cell Death Differ.

Abstract

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3-/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sendai virus and MHV68 cause RIP1- and RIP3-dependent necroptosis. (a) L929 cells were treated with zVAD (10 μM) and infected with MCMV (MOI 5), HSV-1 (MOI 5), LCMV (MOI 5), MHV68 (MOI 5), SeV (10 HA U/ml), KSHV (titre giving 70% infection) or WSN (MOI 5) and % cell survival was determined by the CellTiter-Glo assay. (b) Cell death of L929 cells treated with zVAD (10 μM), Necrostatin-1 (20 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). (c) L929 cells stably expressing mouse shRIP3 or control shRNA were treated with or without zVAD (5 μM) in the absence or presence of SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: cell lysates of the shRNA retrovirally infected cells were probed by RIP3- or actin-specific antibodies. (d) L929 cells were mock-treated or treated with Necrostatin-1 (Nec-1) at 20 μM and infected with indicated titres of SeV. Cell death was monitored 18–20 h post-infection by CellTiter-Glo. (e) Increased relative expression of viral NP protein from RIP3 shRNA knockdown cells as compared to control shRNA knockdown cells by quantitative RT-PCR. Expression is relative to mock-infected samples after normalization to γ-actin. (f) TNF ELISA from supernatants of L929 cells treated with zVAD and infected with SeV (10 or 100 HA U/ml) or MHV68 (MOI 5 or 10). (g) Death of cells treated with control or anti-TNF neutralizing antibody, zVAD (10 μM and 100 μM), TNF (60 pg/ml), SeV (100 HA U/ml) or MHV68 (MOI 5). (h) LA-4 cells were treated with zVAD (20 μM), TNF (100 ng/ml) or Nec-1 (20 μM) and infected with SeV (100 HA U/ml) or MHV68 (MOI 10). Cell survival was monitored 24 h post-infection. (i) LA-4 cells were treated with anti-control or anti-TNF neutralizing antibodies and treated with zVAD, TNF and infected with SeV (100 HA U/ml) before cell survival was assayed. Experiment repeated twice. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments (unless otherwise stated). ***P<0.001, **P<0.01, *P<0.05
Figure 2
Figure 2
Sendai virus induces necroptosis through RIG-I while MHV68 signals through STING. (a) L929 cells stably expressing mouse RIG-I shRNA or control shRNA were treated with zVAD (10 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: knockdown was confirmed by western blot of L929 cells infected with SeV to upregulate RIG-I protein expression. (b) L929 cells stably expressing mouse STING shRNA or control shRNA were treated with zVAD (10 μM) and infected with SeV (10 HA U/ml) or MHV68 (MOI 5). Right panel: knockdown was confirmed by western blot. Data are averages of triplicates from a single experiment, which is representative of at least three independent experiments
Figure 3
Figure 3
Sendai virus Y1/Y2 proteins are required for necroptosis. (a) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). (b) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). (c) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. (d) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ-actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ-actin. Standard error of the mean was calculated from three independent experiments. (e) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. (f) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. ***P<0.001
Figure 4
Figure 4
Loss of RIP1 ubiquitination determines SeV-induced necroptosis. (a) L929 cells were treated as indicated and cell lysates harvested in 1% SDS lysis buffer. RIP1 immunoprecipitation was carried out from SDS lysates and ubiquitination monitored by immunoblot using ubiquitin-specific antibodies. RIP1-specific antibodies were used to ensure uniform RIP1 immunoprecipitation. (b) Infection with d2Y mutant yields higher RIP1 ubiquitination compared to WT SeV infection. A similar experiment as (a) was carried out but using SeV Z wild type (WT) or d2Y mutant virus. (c) 293T cells were transfected with indicated plasmids and lysed using 1% NP40 lysis buffer. Immunoprecipitated RIP1 was resolved by SDS-PAGE and immunoblotted with anti-HA to determine ubiquitination. RIP1 western blot was performed to ensure uniform RIP1 immunoprecipitation. Bottom panel: lysates of the 293T cells transfected with the indicated constructs were probed for RIP1 or Y1/Y2 expression using RIP1 or FLAG-specific antibodies. All experiments repeated at least three times
Figure 5
Figure 5
Sendai virus proteins restrict cIAP1-mediated RIP1 ubiquitination. (a) Knockdown of CYLD protein by siRNA in L929 cells rescues SeV induced necroptosis. Cells transfected with control siRNA or CYLD siRNA were treated with the indicated agent and/or SeV. Right panel: western blot analysis of the control siRNA or CYLD siRNA treated cells using CYLD- or β-actin specific antibodies. (b) Knockdown of CYLD using shRNA in 293T cells overexpressing indicated constructs. RIP1 was immunoprecipitated (RIP1 IP) from 1% NP40 lysates and its ubiquitination was monitored by western blot with ubiquitin-specific antibodies. Lower panel: western blot analysis of lysates with the indicated antibodies. (c) Left panel: treatment of L929 cells with 100 nM SMAC Mimetic (SM) and infection with SeV (10 HA U/ml) causes death of cells. Right panel: SMAC mimetic treatment sensitizes low dose zVAD (2.5 μM) and SeV infection to cell death. (d) RIP1 was immunoprecipitated from 1% NP40 lysates generated from 293T cells transfected with indicated constructs. cIAP1-mediated RIP1 ubiquitination was monitored by western blot. Top panel: RIP1 immunoprecipitates (RIP1 IP) were analysed by western blot analysis using ubiquitin- or RIP1-specific antibodies. Lower panel: lysates from the transfected cells were analysed using the indicated antibodies. Asterisk indicates non-specific band. (e) Top panel: loss of cIAP1 protein expression upon infection with SeV after 10 h post-infection (hpi). Lower panel: lysates from L929 cells treated with indicated agents were resolved by SDS-PAGE and western blot analysis confirmed loss of cIAP1 protein in wild type (WT) SeV-infected samples but not d2Y mutant-infected samples. (f) L929 cells were transfected with pCI or Y1/Y2 constructs and subsequently treated with zVAD (2.5 μM) and SM (100 nM). Cell death was determined by 7AAD+ staining by flow cytometry. For, 5A, 5C, 5F, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. All experiments repeated at least three times. ***P<0.001, **P<0.01, *P<0.05
Figure 6
Figure 6
Lung and BAL analysis of SeV-infected wild type and RIP3-deficient mice. (af) Representative haematoxylin- and eosin- (h & e) stained lung sections of uninfected wild type (a, d), SeV-infected wild type mice (b, e) and SeV-infected RIP3−/− mice (c, f) 7 days post-infection. (g) Increased frequency of anti-SeV CD8+ T cells found in BAL of SeV-infected RIP3−/− mice as compared to control mice 9 days post-infection. Results are representative of three control (Ctl) and 3 RIP3−/− mice infected with SeV or 1 control and 1 RIP3−/− mice infected with PBS. (h) Quantitation of anti-SeV CD8+ T cells from control and RIP3−/− mice. *P<0.05

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