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.A denoviruses (Ads) are ubiquitous pathogens that infect a wide range of vertebrates. Ad infection is generally associated with mild disease, but Ads have been increasingly recognized as significant pathogens in infants, the elderly, and immunocompromised patients (1). Ads have evolved diverse mechanisms to counteract host antiviral responses during infection (2). Successful Ad replication relies on functions provided by early region 4 (E4). 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. Relocalization of TIF-1γ and TFII-I by Ad5 E4-ORF3 results in their proteasomal degradation (12, 13). 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....

Adenoviral strategies to overcome innate cellular responses to infection

“…The E4-ORF3 SUMO target proteins have two distinct fates. One group (e.g., TFII-I and TIF-1c) is further degraded by the ubiquitin-proteasome pathway in the absence of any other viral proteins [77][78][79]. In contrast, degradation of the other group (e.g., Mre11 and Nbs1) is mediated by E1B-55K and E4-ORF6, but not by E4-ORF3.…”

“…A comparative SUMO proteomic study revealed that SUMO conjugation of multiple host proteins increased upon Ad5 E4-ORF3 expression [72]. Biochemical studies using an in vitro SUMO-conjugation assay demonstrated that Ad5 E4-ORF3 functions as a SUMO E3 ligase [77]. Like ubiquitylation, sumoylation is carried out by a cascade of enzymatic steps [76].…”

“…One group (e.g., TFII-I and TIF-1c) is further degraded by the ubiquitin-proteasome pathway in the absence of any other viral proteins [77][78][79]. One group (e.g., TFII-I and TIF-1c) is further degraded by the ubiquitin-proteasome pathway in the absence of any other viral proteins [77][78][79].…”

“…Conjugation of poly-SUMO chains onto substrates is known to serve as a signal for SUMO-targeted ubiquitin ligase-mediated proteasomal degradation [80]. E4-ORF3 leads to a significant increase in SUMO conjugation to TFII-I and TIF-1c, whereas just a slight increase in Mre11 and Nbs1 modification [77,82]. It remains unclear whether Mre11 and Nbs1 are modified by only mono-SUMO, but not by poly-SUMO chains, although E4-ORF3 has been shown to induce their conjugation to both SUMO1 and SUMO2 [75].…”

“…SUMOs may then be transferred to a lysine residue(s) in a substrate protein directly by Ubc9 or using a SUMO E3 ligase. Biochemical studies using an in vitro SUMO-conjugation assay demonstrated that Ad5 E4-ORF3 functions as a SUMO E3 ligase [77].…”