title: TRIM5α requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription.
abstract: TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved.
authors.affiliation: MRC Centre of Medical Molecular Virology, Division of Infection and Immunity, University College London, London, UK.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Fletcher
authors.affiliation: Department of Biochemistry and HSC Core Facilities, University of Utah School of Medicine, Salt Lake City, UT, USA.
authors.forename: Devin E
authors.initials: DE
authors.lastname: Christensen
authors.affiliation: Department of Biochemistry and HSC Core Facilities, University of Utah School of Medicine, Salt Lake City, UT, USA.
authors.forename: Chad
authors.initials: C
authors.lastname: Nelson
authors.affiliation: MRC Centre of Medical Molecular Virology, Division of Infection and Immunity, University College London, London, UK.
authors.forename: Choon Ping
authors.initials: CP
authors.lastname: Tan
authors.affiliation: MRC Centre of Medical Molecular Virology, Division of Infection and Immunity, University College London, London, UK.
authors.forename: Torsten
authors.initials: T
authors.lastname: Schaller
authors.affiliation: Cambridge Institute for Medical Research, Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Lehner
authors.affiliation: Department of Biochemistry and HSC Core Facilities, University of Utah School of Medicine, Salt Lake City, UT, USA.
authors.forename: Wesley I
authors.initials: WI
authors.lastname: Sundquist
authors.affiliation: MRC Centre of Medical Molecular Virology, Division of Infection and Immunity, University College London, London, UK g.towers@ucl.ac.uk.
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Towers
mesh_terms: D000090103:Antiviral Restriction Factors; D002352:Carrier Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D048348:Reverse Transcription / Q000502:physiology*; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
chemical_list: D000090103:Antiviral Restriction Factors; D002352:Carrier Proteins; D034741:RNA, Small Interfering; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin; C510626:UBE2W protein, human; D044763:Ubiquitin-Conjugating Enzymes; C477346:TRIM5 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: TRIM5α; Ube2N; Ube2W; restriction; ubiquitin