Proteomic Analysis of Ubiquitin-Like Posttranslational Modifications Induced by the Adenovirus E4-ORF3 Protein

Abstract

Viruses interact with and regulate many host metabolic pathways in order to advance the viral life cycle and counteract intrinsic and extrinsic antiviral responses. The human adenovirus (Ad) early protein E4-ORF3 forms a unique scaffold throughout the nuclei of infected cells and inhibits multiple antiviral defenses, including a DNA damage response (DDR) and an interferon response. We previously reported that the Ad5 E4-ORF3 protein induces sumoylation of Mre11 and Nbs1, which are essential for the DDR, and their relocalization into E4-ORF3-induced nuclear inclusions is required for this modification to occur. In this study, we sought to analyze a global change in ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, by the Ad5 E4-ORF3 protein and to identify new substrates with these modifications. By a comparative proteome-wide approach utilizing immunoprecipitation/mass spectrometry, we found that Ubl modifications of 166 statistically significant lysine sites in 51 proteins are affected by E4-ORF3, and the proteome of modifications spans a diverse range of cellular functions. Ubl modifications of 92% of these identified sites were increased by E4-ORF3. We further analyzed SUMO3 conjugation of several identified proteins. Our findings demonstrated a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and revealed new SUMO3 substrates induced by E4-ORF3. (1, 2). Therefore, Ad has evolved several mechanisms to inhibit the cellular DDR early after infection. The incoming viral genome is coated with a basic viral core protein that may block recognition of the viral DNA by the cellular DDR machinery at the earliest stages of infection (3). Once Ad early protein synthesis ensues, two distinct mechanisms are employed to inhibit the DDR (1, 2). The Ad5 E1B-55K and E4-ORF6 proteins form an E3 ubiquitin (Ub) ligase complex with cellular proteins cullin 5 (CUL5), Rbx1, and elongins B and C (4, 5). Together, this complex leads to ubiquitin-mediated, proteasome-dependent degradation of cellular sensors of DNA damage, including Mre11, Rad50, and Nbs1 (the MRN complex) (6). Inhibition of cellular sensors of DNA damage blocks downstream signaling events and inhibits both DNA damage repair and cell cycle checkpoint signaling. The Ad5 E4-ORF3 protein sequesters MRN proteins into nuclear inclusions, termed nuclear tracks (7), within infected cell nuclei to inhibit MRN activity (6, 8). E4-ORF3 recruits numerous nuclear proteins into these structures, including promyelocytic leukemia (PML) and other PML-nuclear body (PML-NB)-associ- IMPORTANCE The adenovirus E4-ORF3 protein induces dynamic structural changes in the nuclei of infected cells and counteracts host antiviral responses. One of the mechanisms that accounts for this process is the relocalization and sequestration of cellular proteins into an E4-ORF3 nuclear scaffold, but little is known about how this

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

“…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.…”

“…Our previous study showed that TIF-1γ sumoylation is induced by infection with wild-type Ad5, but not by an E4-ORF3-deficient virus (11). To further verify the role of E4-ORF3, we infected His 6 -SUMO3-expressing HeLa cells (27) with increasing amounts of Ad5-WT or an Ad-CMV-E4-ORF3 expression vector and examined TIF-1γ sumoylation by capturing His 6 -SUMO3 with Ni 2+ -NTA resin (11). SUMO3 conjugation to TIF-1γ was increased depending on the E4-ORF3 expression level ( Fig.…”

“…Mre11 and Nbs1 sumoylation is triggered by E4-ORF3 (8) and these proteins are then targeted for proteasomal degradation by other viral proteins, E1B-55K and E4-ORF6, in association with a cellular ubiquitin E3 ligase complex (6). On the other hand, E4-ORF3 is sufficient to mediate sumoylation and proteasomal degradation of TFII-I and TIF-1γ in infected cells (11)(12)(13). Mre11 and Nbs1 sumoylation is transient (8), whereas TFII-I (13) and TIF-1γ (Fig.…”

“…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.…”