title: TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).
abstract: The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: Melody M H
authors.identifier: 0000-0002-6905-7940
authors.initials: MM
authors.lastname: Li
authors.affiliation: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Zerlina
authors.initials: Z
authors.lastname: Lau
authors.affiliation: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Pamela
authors.initials: P
authors.lastname: Cheung
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: Eduardo G
authors.identifier: 0000-0002-1531-1448
authors.initials: EG
authors.lastname: Aguilar
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: William M
authors.initials: WM
authors.lastname: Schneider
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: Leonia
authors.initials: L
authors.lastname: Bozzacco
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, New York, United States of America.
authors.forename: Henrik
authors.identifier: 0000-0001-8950-4990
authors.initials: H
authors.lastname: Molina
authors.affiliation: Trans-NIH RNAi Screening Facility, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, NIH, Rockville, Maryland, United States of America.
authors.forename: Eugen
authors.initials: E
authors.lastname: Buehler
authors.affiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Takaoka
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Integrated Screening Core, Experimental Therapeutics Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Dan P
authors.initials: DP
authors.lastname: Felsenfeld
authors.affiliation: Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, United States of America.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: MacDonald
mesh_terms: D018354:Alphavirus Infections / Q000517:prevention & control*; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D057809:HEK293 Cells; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism; D000072417:Protein Domains; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012845:Sindbis Virus / Q000254:growth & development*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000070584:Tripartite Motif Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000998:Antiviral Agents; D007370:Interferon Type I; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; C527496:ZC3HAV1 protein, human; C084523:TRIM25 protein, human; D044767:Ubiquitin-Protein Ligases