Full text links CiteDisplay options Display options Format AbstractPubMedPMID
Abstract
Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense. Receptor-interacting protein kinase (RIP) 3 (also called RIPK3) mediates RIP homotypic interaction motif (RHIM)-dependent programmed necrosis induced by murine cytomegalovirus (MCMV) infection or death receptor activation and suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI, also known as ZBP1 or DLM-1) sensitizes cells to virus-induced necrosis and that DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3(-/-) mice, vIRA mutant MCMV pathogenesis is restored in DAI(-/-) mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.
Figure 1. RIP3 levels differentially confer susceptibility to necroptosis and virus-induced programmed necrosis
(A) Immunoblot…
Figure 1. RIP3 levels differentially confer susceptibility to necroptosis and virus-induced programmed necrosis (A) Immunoblot (IB) analysis to detect RIP3, RIP1 and β-actin from NIH3T3 cells stably expressing 3XFLAG-tagged RIP3, RIP3mRHIM, or control. (B) Viability of RIP3-expressing NIH3T3 stable cell lines infected with M45mutRHIM MCMV at a multiplicity of infection (MOI) of 10. (n=4) (C) Viability of RIP3-expressing NIH3T3 stable cell lines after treatment with TNF (25 ng/ml) and zVAD-fmk (50 μM) to induce necroptosis (n=3–4). (D) Viability of NIH3T3, 3T3-SA and SVEC4-10 cell lines infected with M45mutRHIM MCMV (MOI=10). (n=3–7) (E) IB analysis of uninfected NIH3T3, 3T3-SA, and SVEC4-10 cells to detect DAI, RIP3, RIP1, and β-actin. A non-specific (*ns) band is characteristic for this antibody (Karayel et al., 2009). **p<0.001, #not significant (p>0.05). See also related Figure S1.
Figure 2. Increased DAI expression sensitizes resistant…
Figure 2. Increased DAI expression sensitizes resistant cells to virus-induced necrosis
(A) IB analysis to…
Figure 2. Increased DAI expression sensitizes resistant cells to virus-induced necrosis (A) IB analysis to detect FLAG and β-actin of NIH3T3 stable cells expressing FLAG-tagged WT DAI, FLAG-tagged DAImRHIM, or control. (B) Viability of DAI-expressing NIH3T3 stable cell lines infected with M45mutRHIM MCMV (MOI=10) and treated with DMSO or Nec-1 (30 μM). (n=4) (C) IB analysis to detect FLAG, RIP3, RIP1 and β-actin in stable NIH3T3 cell lines expressing FLAG-tagged WT DAI together with either a scramble control (Sc) or one of two RIP3-specific shRNAs (RIP3-A or RIP3-B). (D) Viability of DAI-expressing, RIP3 knockdown NIH3T3 stable cell lines following infection with M45mutRHIM MCMV (MOI=10)(n=3). **p<0.001, #not significant (p>0.05). See also related Figure S2.
Figure 3. DAI and RIP3 cooperate in…
Figure 3. DAI and RIP3 cooperate in virus-induced necrosis
(A) RIP3 and DAI interaction in…
Figure 3. DAI and RIP3 cooperate in virus-induced necrosis (A) RIP3 and DAI interaction in the absence of vIRA. IB of immunoprecipitations (IP) and whole cell lysates (WCL; 5% total) showing DAI, M45, RIP3 and β-actin in 3T3-SA cells infected with M45mutRHIM MCMV (MOI=5) harvested at the indicated times (h.p.i.). (B) IB analysis to detect DAI, RIP3, RIP1 and β-actin in 3T3-SA cells transfected with non-targeting (NT) control, DAI, or RIP3 siRNAs (C) Viability of 3T3-SA cells transfected with the indicated siRNA and infected with M45mutRHIM (MOI=10) and treated with DMSO control or Nec-1 inhibitor (30μM). (n=4) (D) IB analysis to detect DAI, RIP3, RIP1, and β-actin in C57BL/6 control, DAI−/−, and RIP3−/− murine embryonic fibroblasts (MEFs) (E) Viability of C57BL/6 control, DAI−/−, and RIP3−/− MEFs infected with M45mutRHIM MCMV (MOI=10)(n=3). (F) Single-step replication of WT and M45mutRHIM MCMV (MOI of 5) on control C57BL/6 (left) and DAI−/− (right) MEFs. Viral titers determined by plaque assay with the first (0 h) time point representing amount of virus in the inoculum. Error bars, SD. *p<0.01, **p<0.001, #not significant (p>0.05). See also related Figure S3.
Figure 4. M45 mut RHIM virus attenuation…
Figure 4. M45 mut RHIM virus attenuation in vivo is reversed in DAI −/− mice
(A) Footpad swelling…
Figure 4. M45mutRHIM virus attenuation in vivo is reversed in DAI−/− mice (A) Footpad swelling induced by parental WT or M45mutRHIM MCMV infection. Footpad thickness of C57BL/6 control (top) and DAI−/− (bottom) mice inoculated with 106 pfu was measured with a digital caliper, and mean values plotted at the indicated times over a 14 day time course (n=7–9 animals/group). (B–D) Viral titers from spleen (B), liver (C), and SGs (D) of control (C57BL/6), RIP3−/−, and DAI−/− mice infected via intraperitoneal inoculation with 106 pfu of indicated virus and harvested at the indicated time (n= 5–10 animals/group). Error bars, SEM. See also related Figure S4. See this image and copyright information in PMC
Comment in
A way to DAI. Welz PS, Pasparakis M.Welz PS, et al.Cell Host Microbe. 2012 Mar 15;11(3):223-5. doi: 10.1016/j.chom.2012.02.003.Cell Host Microbe. 2012.PMID: 22423962
DAI/ZBP1/DLM-1 Complexes with RIP3 to Mediate Virus-Induced Programmed Necrosis that Is Targeted by Murine Cytomegalovirus vIRA. Upton JW, Kaiser WJ, Mocarski ES.Upton JW, et al.Cell Host Microbe. 2019 Oct 9;26(4):564. doi: 10.1016/j.chom.2019.09.004.Cell Host Microbe. 2019.PMID: 31600504No abstract available.
Similar articles
Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1. Guo H, Gilley RP, Fisher A, Lane R, Landsteiner VJ, Ragan KB, Dovey CM, Carette JE, Upton JW, Mocarski ES, Kaiser WJ.Guo H, et al.Cell Death Dis. 2018 Jul 26;9(8):816. doi: 10.1038/s41419-018-0868-3.Cell Death Dis. 2018.PMID: 30050136Free PMC article.
Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, Sehon CA, Marquis RW, Bertin J, Mocarski ES.Kaiser WJ, et al.J Biol Chem. 2013 Oct 25;288(43):31268-79. doi: 10.1074/jbc.M113.462341. Epub 2013 Sep 9.J Biol Chem. 2013.PMID: 24019532Free PMC article.
DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB. Rebsamen M, Heinz LX, Meylan E, Michallet MC, Schroder K, Hofmann K, Vazquez J, Benedict CA, Tschopp J.Rebsamen M, et al.EMBO Rep. 2009 Aug;10(8):916-22. doi: 10.1038/embor.2009.109. Epub 2009 Jul 10.EMBO Rep. 2009.PMID: 19590578Free PMC article.
Manipulation of apoptosis and necroptosis signaling by herpesviruses. Guo H, Kaiser WJ, Mocarski ES.Guo H, et al.Med Microbiol Immunol. 2015 Jun;204(3):439-48. doi: 10.1007/s00430-015-0410-5. Epub 2015 Apr 1.Med Microbiol Immunol. 2015.PMID: 25828583Free PMC article.Review.
Necroptosis: The Trojan horse in cell autonomous antiviral host defense. Mocarski ES, Guo H, Kaiser WJ.Mocarski ES, et al.Virology. 2015 May;479-480:160-6. doi: 10.1016/j.virol.2015.03.016. Epub 2015 Mar 24.Virology. 2015.PMID: 25819165Free PMC article.Review.
See all similar articles
Cited by
The role of inflammatory response and metabolic reprogramming in sepsis-associated acute kidney injury: mechanistic insights and therapeutic potential. Liu AB, Tan B, Yang P, Tian N, Li JK, Wang SC, Yang LS, Ma L, Zhang JF.Liu AB, et al.Front Immunol. 2024 Oct 31;15:1487576. doi: 10.3389/fimmu.2024.1487576. eCollection 2024.Front Immunol. 2024.PMID: 39544947Free PMC article.Review.
The Evolution and Biological Activity of Metazoan Mixed Lineage Kinase Domain-Like Protein (MLKL). Wang Q, Yuan Z, Xu H, Chen Y, Sun L.Wang Q, et al.Int J Mol Sci. 2024 Oct 2;25(19):10626. doi: 10.3390/ijms251910626.Int J Mol Sci. 2024.PMID: 39408954Free PMC article.
RIPK1 is essential for Herpes Simplex Virus-triggered ZBP1-dependent necroptosis in human cells. Amusan OT, Wang S, Yin C, Koehler HS, Li Y, Tenev T, Wilson R, Bellenie B, Zhang T, Wang J, Liu C, Seong K, Poorbaghi SL, Yates J, Shen Y, Upton JW, Meier P, Balachandran S, Guo H.Amusan OT, et al.bioRxiv [Preprint]. 2024 Sep 19:2024.09.17.613393. doi: 10.1101/2024.09.17.613393.bioRxiv. 2024.PMID: 39345610Free PMC article.Preprint.
A shorter splicing isoform antagonizes ZBP1 to modulate cell death and inflammatory responses. Nagata M, Carvalho Schäfer Y, Wachsmuth L, Pasparakis M.Nagata M, et al.EMBO J. 2024 Nov;43(21):5037-5056. doi: 10.1038/s44318-024-00238-7. Epub 2024 Sep 19.EMBO J. 2024.PMID: 39300211Free PMC article.
No Time to Die: How Cytomegaloviruses Suppress Apoptosis, Necroptosis, and Pyroptosis. Deng Y, Águeda-Pinto A, Brune W.Deng Y, et al.Viruses. 2024 Aug 9;16(8):1272. doi: 10.3390/v16081272.Viruses. 2024.PMID: 39205246Free PMC article.Review.
Benedict CA, Angulo A, Patterson G, Ha S, Huang H, Messerle M, Ware CF, Ghazal P. Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro. J Virol. 2004;78:741–750. - PMC - PubMed
Best SM. Viral subversion of apoptotic enzymes: escape from death row. Annual review of microbiology. 2008;62:171–192. - PMC - PubMed
Brune W, Menard C, Heesemann J, Koszinowski UH. A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism. Science. 2001;291:303–305. - PubMed
Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, Yang X, Iotzova VS, Clarke W, Strnad J, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem. 2003;278:1450–1456. - PubMed
Show all 43 references
Publication types
Research Support, N.I.H., Extramural Actions
Search in PubMed
Search in MeSH
Add to Search
MeSH terms
Animals Actions
Search in PubMed
Search in MeSH
Add to Search
Apoptosis Actions
Search in PubMed
Search in MeSH
Add to Search
Glycoproteins / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Glycoproteins / physiology* Actions
Search in PubMed
Search in MeSH
Add to Search
Herpesviridae Infections / immunology* Actions
Search in PubMed
Search in MeSH
Add to Search
Herpesviridae Infections / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Herpesviridae Infections / pathology Actions
Search in PubMed
Search in MeSH
Add to Search
Host-Pathogen Interactions Actions
Search in PubMed
Search in MeSH
Add to Search
Inflammation / immunology Actions
Search in PubMed
Search in MeSH
Add to Search
Inflammation / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Inflammation / virology Actions
Search in PubMed
Search in MeSH
Add to Search
Interferon-beta / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Mice Actions
Search in PubMed
Search in MeSH
Add to Search
Mice, Inbred C57BL Actions
Search in PubMed
Search in MeSH
Add to Search
Mice, Knockout Actions
Search in PubMed
Search in MeSH
Add to Search
Multiprotein Complexes / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Multiprotein Complexes / physiology Actions
Search in PubMed
Search in MeSH
Add to Search
Muromegalovirus / genetics Actions
Search in PubMed
Search in MeSH
Add to Search
Muromegalovirus / immunology Actions
Search in PubMed
Search in MeSH
Add to Search
Muromegalovirus / physiology* Actions
Search in PubMed
Search in MeSH
Add to Search
NF-kappa B / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
NIH 3T3 Cells Actions
Search in PubMed
Search in MeSH
Add to Search
Necrosis / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Necrosis / virology* Actions
Search in PubMed
Search in MeSH
Add to Search
RNA-Binding Proteins Actions
Search in PubMed
Search in MeSH
Add to Search
Receptor-Interacting Protein Serine-Threonine Kinases / genetics Actions
Search in PubMed
Search in MeSH
Add to Search
Receptor-Interacting Protein Serine-Threonine Kinases / metabolism* Actions
Search in PubMed
Search in MeSH
Add to Search
Signal Transduction Actions
Search in PubMed
Search in MeSH
Add to Search
Viral Load Actions
Search in PubMed
Search in MeSH
Add to Search
Viral Proteins / genetics Actions
Search in PubMed
Search in MeSH
Add to Search
Viral Proteins / metabolism Actions
Search in PubMed
Search in MeSH
Add to Search
Substances
Glycoproteins Actions
Search in PubMed
Search in MeSH
Add to Search
Multiprotein Complexes Actions
Search in PubMed
Search in MeSH
Add to Search
NF-kappa B Actions
Search in PubMed
Search in MeSH
Add to Search
RNA-Binding Proteins Actions
Search in PubMed
Search in MeSH
Add to Search
Viral Proteins Actions
Search in PubMed
Search in MeSH
Add to Search
Zbp1 protein, mouse Actions
Search in PubMed
Search in MeSH
Add to Search
Interferon-beta Actions
Search in PubMed
Search in MeSH
Add to Search
Receptor-Interacting Protein Serine-Threonine Kinases Actions
