Article TRIM5a requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription

TRIM5a is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their prema-ture dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (poly-Ub) chains to stimulate innate immune responses. Here, we show that TRIM5a employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5a auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5a. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodi-meric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5a internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead aN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5a ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the step-wise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5a restriction mechanism and identify the E2 Ub-conjugating cofactors involved