Adenovirus Regulates Sumoylation of Mre11-Rad50-Nbs1 Components through a Paralog-Specific Mechanism

Abstract

<jats:p>The Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the DNA damage response, presenting challenges for DNA viruses and retroviruses. To inactivate this complex, adenovirus (Ad) makes use of the E1B-55K and E4-open reading frame 6 (ORF6) proteins for ubiquitin (Ub)-mediated, proteasome-dependent degradation of MRN and the E4-ORF3 protein for relocalization and sequestration of MRN within infected-cell nuclei. Here, we report that Mre11 is modified by the Ub-related modifier SUMO-2 and Nbs1 is modified by both SUMO-1 and SUMO-2. We found that Mre11 and Nbs1 are sumoylated during Ad5 infection and that the E4-ORF3 protein is necessary and sufficient to induce SUMO conjugation. Relocalization of Mre11 and Nbs1 into E4-ORF3 nuclear tracks is required for this modification to occur. E4-ORF3-mediated SUMO-1 conjugation to Nbs1 and SUMO-2 conjugation to Mre11 and Nbs1 are transient during wild-type Ad type 5 (Ad5) infection. In contrast, SUMO-1 conjugation to Nbs1 is stable in cells infected with E1B-55K or E4-ORF6 mutant viruses, suggesting that Ad regulates paralog-specific desumoylation of Nbs1. Inhibition of viral DNA replication blocks deconjugation of SUMO-2 from Mre11 and Nbs1, indicating that a late-phase process is involved in Mre11 and Nbs1 desumoylation. Our results provide direct evidence of Mre11 and Nbs1 sumoylation induced by the Ad5 E4-ORF3 protein and an important example showing that modification of a single substrate by both SUMO-1 and SUMO-2 is regulated through distinct mechanisms. Our findings suggest how E4-ORF3-mediated relocalization of the MRN complex influences the cellular DNA damage response.</jats:p>

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation

“…In addition to sumoylation, E4-ORF3 decreased TIF-1γ and TFII-I, but not Nbs1, expression levels (Fig. 1B), consistent with previous reports (8,12). Additionally, we confirmed that E4-ORF3 mediates proteasomal degradation of TIF-1γ in HeLa and A549 cells using the proteasome inhibitor MG132 (Fig.…”

“…2A), demonstrating that TIF-1γ sumoylation was enhanced by E4-ORF3 in the absence of any cellular SUMO E3 ligase. We also tested the E4-ORF3 L103A mutant, which is defective in formation of nuclear tracks and subsequent E4-ORF3 functions, including the sumoylation of target proteins (7,8). Interestingly, TIF-1γ sumoylation remained unchanged by addition of E4-ORF3 L103A in the same range of concentrations as wild-type, suggesting that E4-ORF3 self-assembly is required for this activity.…”

“…Ad5 E4-ORF3 induces sumoylation of multiple cellular proteins (8,11,13). It remained unclear whether E4-ORF3 is a SUMO E3 ligase or employs a cellular E3 ligase.…”

“…We previously showed that Ad5 E4-ORF3 mediates sumoylation of multiple cellular proteins (8,11,13) and suggested that E4-ORF3-induced sumoylation triggers ubiquitination and proteasomal degradation of some substrates. Relocalization of target proteins into E4-ORF3 nuclear tracks is required for substrate sumoylation and proteasomal degradation (8,11,13).…”

“…The highly conserved E4-ORF3 protein assembles into a multimeric nuclear network, referred to as tracks (3), in infected cells (4,5). The Ad5 E4-ORF3 protein recruits numerous cellular proteins into nuclear tracks including promylocytic leukemia (PML) nuclear body components (3), the Mre11-Rad50-Nbs1 (MRN) complex (6,7), small ubiquitin-like modifier (SUMO) proteins (8), transcription intermediary factor (TIF)-1α (9), TIF-1γ (10), and TFII-I (11). This event causes sequestration of the target proteins inhibiting their antiviral functions.…”