{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T14:55:08Z","timestamp":1777560908576,"version":"3.51.4"},"reference-count":39,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,10]],"date-time":"2026-01-10T00:00:00Z","timestamp":1768003200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R21AI135709-02"],"award-info":[{"award-number":["R21AI135709-02"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100004917","name":"Cancer Prevention and Research Institute of Texas","doi-asserted-by":"publisher","award":["R1202"],"award-info":[{"award-number":["R1202"]}],"id":[{"id":"10.13039\/100004917","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Auburn University institutional funds"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Necroptosis is an antiviral form of programmed cell death modulated by proteins that interact via RIP Homotypic Interaction Motifs (RHIMs). The result of the signaling pathways depends on which RHIM-containing proteins are involved: although both host and viral proteins contain RHIMs, virally encoded RHIM proteins, such as murine cytomegalovirus (MCMV)-encoded viral inhibitor of RIP activation (vIRA) serve to prevent cell death. Although every RHIM contains the same core four-amino-acid pattern, there are variations in individual sequences that we hypothesized would determine the differential outcomes in necroptotic signaling. As such, we replaced the RHIM in vIRA with the RHIMs from other proteins involved in the signaling cascade (RIPK1, RIPK3, ZBP1, ICP6) to assess the effect on necroptosis during MCMV infection. Although these RHIM-swap vIRA constructs remained able to bind to RIPK3, in the context of MCMV infection, they lost the ability to prevent necroptosis. These results are consistent with other studies that demonstrate that RHIM-containing proteins form amyloid fibrils unique to the proteins interfacing. Our results provide biological context for the growing model that the outcome of RHIM-based signaling is influenced by the specific amyloid fibril structures that are driven by the unique amino-acid sequences of each RHIM present.<\/jats:p>","DOI":"10.3390\/pathogens15010079","type":"journal-article","created":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T09:13:01Z","timestamp":1768209181000},"page":"79","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["vIRA Inhibition of Antiviral Necroptosis and RIPK3 Binding Are Separable Events"],"prefix":"10.3390","volume":"15","author":[{"given":"Katherine B.","family":"Ragan","sequence":"first","affiliation":[{"name":"Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, TX 78712, USA"},{"name":"Independent Researcher, Atlanta, GA 30317, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haripriya","family":"Sridharan","sequence":"additional","affiliation":[{"name":"Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, TX 78712, USA"},{"name":"Thermo Fisher Scientific, Whitefield, Bangalore 560066, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aaron S.","family":"Stark","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA"},{"name":"Independent Researcher, New Braunfels, TX 78130, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaela","family":"Ilami","sequence":"additional","affiliation":[{"name":"Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, TX 78712, USA"},{"name":"Independent Researcher, Austin, TX 78724, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2634-144X","authenticated-orcid":false,"given":"Amanda D.","family":"Fisher","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health at San Antonio, San Antonio, TX 78229, USA"},{"name":"Independent Researcher, Atlanta, GA 30052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1101-274X","authenticated-orcid":false,"given":"Olivia N.","family":"Brahms","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William J.","family":"Kaiser","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health at San Antonio, San Antonio, TX 78229, USA"},{"name":"Independent Researcher, Sylvania, GA 30467, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4218-4026","authenticated-orcid":false,"given":"Jason W.","family":"Upton","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.tim.2014.01.005","article-title":"Programmed Necrosis in Microbial Pathogenesis","volume":"22","author":"Sridharan","year":"2014","journal-title":"Trends Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.semcdb.2014.07.013","article-title":"Necroptosis in Health and Diseases","volume":"35","author":"Zhou","year":"2014","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1016\/j.virol.2015.03.016","article-title":"Necroptosis: The Trojan Horse in Cell Autonomous Antiviral Host Defense","volume":"479\u2013480","author":"Mocarski","year":"2015","journal-title":"Virology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1112","DOI":"10.1016\/j.cell.2009.05.037","article-title":"Phosphorylation-Driven Assembly of the RIP1-RIP3 Complex Regulates Programmed Necrosis and Virus-Induced Inflammation","volume":"137","author":"Cho","year":"2009","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"16871","DOI":"10.1074\/jbc.274.24.16871","article-title":"RIP3, a Novel Apoptosis-Inducing Kinase","volume":"274","author":"Sun","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e960759","DOI":"10.4161\/23723548.2014.960759","article-title":"Execution of RIPK3-Regulated Necrosis","volume":"1","author":"Cai","year":"2014","journal-title":"Mol. Cell Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1038\/ncb2883","article-title":"Plasma Membrane Translocation of Trimerized MLKL Protein Is Required for TNF-Induced Necroptosis","volume":"16","author":"Cai","year":"2014","journal-title":"Nat. Cell Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.cell.2011.11.031","article-title":"Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase","volume":"148","author":"Sun","year":"2012","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5322","DOI":"10.1073\/pnas.1200012109","article-title":"Mixed Lineage Kinase Domain-like Is a Key Receptor Interacting Protein 3 Downstream Component of TNF-Induced Necrosis","volume":"109","author":"Zhao","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1038\/cr.2013.171","article-title":"Translocation of Mixed Lineage Kinase Domain-like Protein to Plasma Membrane Leads to Necrotic Cell Death","volume":"24","author":"Chen","year":"2014","journal-title":"Cell Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1038\/cr.2013.91","article-title":"Mlkl Knockout Mice Demonstrate the Indispensable Role of Mlkl in Necroptosis","volume":"23","author":"Wu","year":"2013","journal-title":"Cell Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.molcel.2014.03.003","article-title":"Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption upon Phosphorylation by RIP3","volume":"54","author":"Wang","year":"2014","journal-title":"Mol. Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4942","DOI":"10.4049\/jimmunol.174.8.4942","article-title":"Apoptosis Induced by the Toll-like Receptor Adaptor TRIF Is Dependent on Its Receptor Interacting Protein Homotypic Interaction Motif","volume":"174","author":"Kaiser","year":"2005","journal-title":"J. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"31268","DOI":"10.1074\/jbc.M113.462341","article-title":"Toll-like Receptor 3-Mediated Necrosis via TRIF, RIP3, and MLKL","volume":"288","author":"Kaiser","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1111\/imr.12539","article-title":"RIPK3-Driven Cell Death during Virus Infections","volume":"277","author":"Upton","year":"2017","journal-title":"Immunol. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Steain, M., Baker, M.O.D.G., Pham, C.L.L., Shanmugam, N., Gambin, Y., Sierecki, E., McSharry, B.P., Avdic, S., Slobedman, B., and Sunde, M. (2020). Varicella Zoster Virus Encodes a Viral Decoy RHIM to Inhibit Cell Death. PLoS Pathog., 16.","DOI":"10.1101\/2020.03.12.988659"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6427","DOI":"10.4049\/jimmunol.181.9.6427","article-title":"Receptor-Interacting Protein Homotypic Interaction Motif-Dependent Control of NF-Kappa B Activation via the DNA-Dependent Activator of IFN Regulatory Factors","volume":"181","author":"Kaiser","year":"2008","journal-title":"J. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1038\/embor.2009.109","article-title":"DAI\/ZBP1 Recruits RIP1 and RIP3 through RIP Homotypic Interaction Motifs to Activate NF-kappaB","volume":"10","author":"Rebsamen","year":"2009","journal-title":"EMBO Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"16966","DOI":"10.1074\/jbc.C800051200","article-title":"Cytomegalovirus M45 Cell Death Suppression Requires Receptor-Interacting Protein (RIP) Homotypic Interaction Motif (RHIM)-Dependent Interaction with RIP1","volume":"283","author":"Upton","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1007\/s00430-015-0410-5","article-title":"Manipulation of Apoptosis and Necroptosis Signaling by Herpesviruses","volume":"204","author":"Guo","year":"2015","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1038\/s41419-018-0868-3","article-title":"Species-Independent Contribution of ZBP1\/DAI\/DLM-1-Triggered Necroptosis in Host Defense against HSV1","volume":"9","author":"Guo","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1126\/science.291.5502.303","article-title":"A Ribonucleotide Reductase Homolog of Cytomegalovirus and Endothelial Cell Tropism","volume":"291","author":"Brune","year":"2001","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.tibs.2008.09.008","article-title":"Tinkering with a Viral Ribonucleotide Reductase","volume":"34","author":"Lembo","year":"2009","journal-title":"Trends Biochem. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"15438","DOI":"10.1073\/pnas.1412767111","article-title":"Direct Activation of RIP3\/MLKL-Dependent Necrosis by Herpes Simplex Virus 1 (HSV-1) Protein ICP6 Triggers Host Antiviral Defense","volume":"111","author":"Wang","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.chom.2015.01.002","article-title":"RIP1\/RIP3 Binding to HSV-1 ICP6 Initiates Necroptosis to Restrict Virus Propagation in Mice","volume":"17","author":"Huang","year":"2015","journal-title":"Cell Host Microbe"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9505","DOI":"10.1074\/jbc.M109488200","article-title":"Identification of a Novel Homotypic Interaction Motif Required for the Phosphorylation of Receptor-Interacting Protein (RIP) by RIP3","volume":"277","author":"Sun","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1244","DOI":"10.1016\/j.cell.2018.03.032","article-title":"The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex","volume":"173","author":"Li","year":"2018","journal-title":"Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e46518","DOI":"10.15252\/embr.201846518","article-title":"Viral M45 and Necroptosis-Associated Proteins Form Heteromeric Amyloid Assemblies","volume":"20","author":"Pham","year":"2019","journal-title":"EMBO Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1038\/s41418-020-0598-9","article-title":"RIP3-Mediated Necroptosis Is Regulated by Inter-Filament Assembly of RIP Homotypic Interaction Motif","volume":"28","author":"Hu","year":"2021","journal-title":"Cell Death Differ."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e2022933118","DOI":"10.1073\/pnas.2022933118","article-title":"The Structure of a Minimum Amyloid Fibril Core Formed by Necroptosis-Mediating RHIM of Human RIPK3","volume":"118","author":"Wu","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1038\/s41467-021-21881-2","article-title":"The Amyloid Structure of Mouse RIPK3 (Receptor Interacting Protein Kinase 3) in Cell Necroptosis","volume":"12","author":"Wu","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6975","DOI":"10.1038\/s41467-024-51303-y","article-title":"The Structure of Mouse RIPK1 RHIM-Containing Domain as a Homo-Amyloid and in RIPK1\/RIPK3 Complex","volume":"15","author":"Liu","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1016\/j.chom.2010.03.006","article-title":"Virus Inhibition of RIP3-Dependent Necrosis","volume":"7","author":"Upton","year":"2010","journal-title":"Cell Host Microbe"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1429","DOI":"10.15252\/embr.201743947","article-title":"Murine Cytomegalovirus IE3-Dependent Transcription Is Required for DAI\/ZBP1-Mediated Necroptosis","volume":"18","author":"Sridharan","year":"2017","journal-title":"EMBO Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"9433","DOI":"10.1128\/JVI.00533-08","article-title":"Control of Cytoplasmic Maturation Events by Cytomegalovirus Tegument Protein Pp150","volume":"82","author":"Tandon","year":"2008","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2998","DOI":"10.1128\/JVI.79.5.2998-3008.2005","article-title":"Use of a Murine Cytomegalovirus K181-Derived Bacterial Artificial Chromosome as a Vaccine Vector for Immunocontraception","volume":"79","author":"Redwood","year":"2005","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4278","DOI":"10.1128\/JVI.78.8.4278-4288.2004","article-title":"The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis","volume":"78","author":"Lembo","year":"2004","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"26642","DOI":"10.1074\/jbc.M114.589515","article-title":"PARP12, an Interferon-Stimulated Gene Involved in the Control of Protein Translation and Inflammation","volume":"289","author":"Welsby","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.chom.2012.01.016","article-title":"DAI\/ZBP1\/DLM-1 Complexes with RIP3 to Mediate Virus-Induced Programmed Necrosis That Is Targeted by Murine Cytomegalovirus vIRA","volume":"11","author":"Upton","year":"2012","journal-title":"Cell Host Microbe"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/79\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T05:16:16Z","timestamp":1768281376000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/1\/79"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,10]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["pathogens15010079"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15010079","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,10]]}}}