TRIM5α requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription.

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.

Fletcher AJ ，Christensen DE ，Nelson C ，Tan CP ，Schaller T ，Lehner PJ ，Sundquist WI ，Towers GJ ... -  《-》

Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling.

TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5's antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform.

Fletcher AJ ，Vaysburd M ，Maslen S ，Zeng J ，Skehel JM ，Towers GJ ，James LC ... -  《-》

A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligases.

Kiss L ，Zeng J ，Dickson CF ，Mallery DL ，Yang JC ，McLaughlin SH ，Boland A ，Neuhaus D ，James LC ... -  《Nature Communications》

Ubiquitination of E3 ubiquitin ligase TRIM5 alpha and its potential role.

Yamauchi K ，Wada K ，Tanji K ，Tanaka M ，Kamitani T ... -  《FEBS Journal》

K63-Linked Ubiquitin Is Required for Restriction of HIV-1 Reverse Transcription and Capsid Destabilization by Rhesus TRIM5α.

TRIM5α is an antiviral restriction factor that inhibits retroviral infection in a species-specific fashion. TRIM5α binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin-dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5α and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5α and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5α. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5α with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5α is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5α with autophagosomal membranes and the autophagic adapter protein p62.IMPORTANCE Although the mechanisms by which TRIM5α can induce the abortive disassembly of retroviral capsids have remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage-specific deubiquitinating enzymes tethered to TRIM5α, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation by TRIM5α, providing evidence for a noncanonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5α and potentially providing insight into the mechanism of action of other TRIM family proteins.

Imam S ，Kömürlü S ，Mattick J ，Selyutina A ，Talley S ，Eddins A ，Diaz-Griffero F ，Campbell EM ... -  《-》