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

TRIM5 alpha 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 TRIM5 alpha employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5 alpha auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5 alpha. 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 TRIM5 alpha internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead alpha N-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5 alpha 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 TRIM5 alpha restriction mechanism and identify the E2 Ub-conjugating cofactors involved.