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

Abstract

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.

Keywords: E3 ligase; SUMO; TIF-1γ; adenovirus; proteasome degradation.

Fig. 1.

E4-ORF3 enhances TIF-1γ sumoylation and proteasomal degradation. (A) His₆-tagged SUMO3-expressing HeLa cells were infected with increasing amounts of wild-type Ad5 [Ad5-WT, 0, 50, 100, 200, and 400 virus paticles per cell (P/cell)] and SUMO-conjugated proteins were analyzed at 8 hours post-infection (hpi) (pull-down). RanGAP1 was used as a control for SUMO capture. Total cell lysates were analyzed by Western blot (lysate). (B) Recombinant empty Ad-CMV (lanes 1 and 2, 900 P/cell) or Ad-CMV-HA-E4-ORF3 expression vector (lanes 3–5, 100, 300, and 900 P/cell) were used to infect His₆-SUMO3-HeLa cells. At 10 hpi, SUMO conjugates were analyzed as in A. (C) HeLa and A549 cells were infected with empty Ad-CMV (1,000 P/cell) or Ad-CMV-HA-E4-ORF3 (200 and 1,000 P/cell) and treated with 20 μM MG132 at 1 hpi. At 10 hpi (HeLa) or 12 hpi (A549), cells were harvested and protein levels were determined by Western blot.

Fig. 2.

In vitro sumoylation of TIF-1γ with E4-ORF3. (A) GST-tagged recombinant TIF-1γ protein (100 nM) was incubated with 50 nM E1, 250 nM E2, 50 μM His₆-SUMO3, and the indicated concentrations of His₆-E4-ORF3-WT or His₆-E4-ORF3-L103A proteins at 37 °C for 60 min. Reaction mixtures were analyzed by Western blot with anti–TIF-1γ, anti-SUMO2/3, and anti–E4-ORF3 antibodies. (B) GST–TIF-1γΔC (100 nM) was used as a substrate and sumoylation was analyzed as described in A. (C) GST–TIF-1γΔC was incubated with (+WT and +L103A) or without (−) His₆–E4-ORF3 for the indicated time periods. (D) GST–TIF-1γΔC was sumoylated at 37 °C for 60 min and incubated with 10 nM SENP1 catalytic domain at 20 °C for 10 min. A total of 3 μM His₆–E4-ORF3 was used in C and D. (E) Schematic representation of TIF-1γ and TIF-1γΔC. RBCC contains the RING finger, B box, and coiled-coil domain; PB contains the plant homeodomain (PHD) and bromodomain.

Fig. S1.

In vitro sumoylation of TIF-1γ with E4-ORF3. GST–TIF-1γΔC (100 nM) was incubated with 50 nM E1, 250 nM E2, 50 μM His₆-SUMO3, and the indicated concentrations of His₆–E4-ORF3-WT or His₆–E4-ORF3-L103A proteins at 37 °C for 90 min. Reaction mixtures were analyzed by Western blot with anti–TIF-1γ, anti-SUMO2/3, and anti–E4-ORF3 antibodies.

Fig. 3.

Poly-SUMO3 chain assembly with E4-ORF3. (A) The indicated concentrations of His₆–E4-ORF3-WT or His₆–E4-ORF3-L103A proteins were added to reaction mixtures described in Fig. 2A and incubated at 37 °C for 60 min. Products were analyzed by Western blot with anti-SUMO2/3 and anti–E4-ORF3 antibodies. The two images in the panel separated by a white space were obtained from the same source file. (B) The E1/E2/His₆-SUMO3 mixture was incubated with (+E4-ORF3) or without (−) His₆–E4-ORF3 for the indicated time periods and products were analyzed by Western blot with anti-SUMO2/3 antibody. (C) E1/E2/His₆-SUMO3 and His₆–E4-ORF3 were incubated at 37 °C for 60 min and further incubated with 10 nM SENP1 catalytic domain at 20 °C for 10 min. (D) E1 or E2 enzyme was left out of the reaction mixtures described in Fig. 2A, incubated at 37 °C for 60 min. A total of 1.5 μM His₆–E4-ORF3 was used in B–D.

Fig. 4.

In vitro sumoylation assays with SUMO3 (K11R). (A) GST–TIF-1γΔC was incubated in reaction mixtures described in Fig. 2A with 10 μM of His₆-SUMO3 or His₆-SUMO3 (K11R) at 37 °C for 60 min or 180 min in the presence or absence of 3 μM His₆–E4-ORF3. (B) Poly-SUMO chain formation reactions were performed as described in Fig. 2A using 50 μM SUMO proteins and 1.5 μM His₆–E4-ORF3 at 37 °C for 60 min or 180 min.

Fig. 5.

Interaction between E4-ORF3 and SUMO machinery proteins. (A) HeLa cells were transfected with a Myc-Ubc9 expression vector and infected with a recombinant empty Ad-CMV, Ad-CMV-HA-E4-ORF3-WT, or Ad-CMV-HA-E4-ORF3-L103A expression vector. At 24 hpi, cells were lysed and Myc-Ubc9 was immunoprecipitated using anti-Myc antibody or control IgG. Protein levels were analyzed by Western blot. (B) His₆–SUMO3-expressing HeLa cells were left uninfected or infected with dl355 (ΔE4-ORF6), dl355/inORF3 (ΔE4-ORF6/ΔE4-ORF3), dl355/L103A (ΔE4-ORF6/E4-ORF3 point mutant L103A), or dl355/DL (ΔE4-ORF6/ E4-ORF3 point mutant D105A/L106A) for 8 h. SUMO conjugates were analyzed as described in Fig. 1A. (C) HeLa cells were infected with the indicated viruses for 8 h. E4-ORF3 and TIF-1γ were immunostained with anti–E4-ORF3 and anti–TIF-1γ antibodies and visualized by fluorescence microscopy.

Fig. 6.

In vitro STUbL assay. (A) Mixtures of di- to octa-SUMO3 chains (50 ng) were incubated with 5 μM HA-ubiquitin, 100 nM His₆-UBE1, and 200 nM UbcH5a in the presence of 0.5 or 2.5 μM His₆–E4-ORF3 or 0.5 μM His₆-RNF4 at 37 °C for 90 min. Ubiquitination of SUMO chains was analyzed by Western blot using anti-SUMO2/3 and anti-ubiquitin antibodies. Input His₆-tagged proteins were visualized using anti-His antibody. (B) His₆–E4-ORF3, His₆-RNF4, or BSA was incubated with recombinant poly-SUMO chains and pulled down using Ni²⁺-NTA beads. Bound proteins were analyzed by Western blot with antibodies against SUMO2/3, RNF4, and E4-ORF3. A total of 5% of the input reaction is shown in lane 1.