TRIM5α is a SUMO substrate

Background: The TRIM5 alpha restriction factor interferes with retroviral infections by inhibiting an early step of viral replication. TRIM5 alpha activity was recently proposed to be regulated by the SUMO machinery and one SUMO consensus conjugation site as well as three putative SUMO interacting motifs (SIMs) were identified within TRIM5 alpha sequence. Whereas mutation of the SIM sequences was found to abolish TRIM5 alpha antiviral activity, mutation of the consensus SUMO conjugation site did not affect its restriction capacity, although this putative site has never been shown to be actually a SUMO substrate. Findings: Here we further demonstrate that TRIM5 alpha relies on the SUMO machinery to promote restriction, since SUMO1 overexpression enhances TRIM5 alpha-mediated retroviral inhibition whereas knockdown of SUMO1 or E2 SUMO conjugating enzyme Ubc9 prevents restriction. Furthermore, we show for the first time that TRIM5 alpha is SUMOylated both in vitro and in cellulo and that Lysine 10 is the main SUMOylation site. Mutation of the consensus SUMO conjugation motif in position 10 abrogated SUMOylation at this position, but did not disrupt TRIM5 alpha antiviral activity. Conclusions: Altogether, our results confirm that the SUMO machinery is involved in TRIM5 alpha-mediated retroviral restriction, and demonstrate that TRIM5 alpha is a SUMO 1 and SUMO 2 substrate. The inability to abrogate TRIM5 alpha antiviral activity by mutating its main SUMO conjugation motif supports the notion that non-covalent interaction with SUMO or SUMOylated proteins rather than TRIM5 alpha direct SUMOylation is required.