The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

Abstract

Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection.

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.…”

“…The SUMO E4 elongase ZNF451 uses tandem SIMs for activity (30). The predicted E4-ORF3 SIM contains the L103 residue, which is crucial for E4-ORF3 polymer assembly (7), and residues D105/L106, which are not required for E4-ORF3 self-assembly and sequestration of PML and SUMOs, but are absolutely required for relocalization of the MRN complex and TFII-I (7,8,13). We examined whether the predicted SIM in E4-ORF3 is critical for sumoylation of TIF-1γ using the in vivo sumoylation assay.…”

“…1) (12). We recently showed that E4-ORF3 targets TFII-I for sumoylation and proteasomal degradation (13). However, it remained unclear whether E4-ORF3 itself is a STUbL or uses a cellular STUbL for degradation of sumoylated proteins.…”

“…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.…”

“…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).…”