The Adenovirus E4 ORF3 Protein Binds and Reorganizes the TRIM Family Member Transcriptional Intermediary Factor 1 Alpha

Abstract

One of the most interesting functions attributed to the adenovirus early region 4 open reading frame 3 (E4 ORF3) protein is its reorganization of promyelocytic leukemia (PML) protein nuclear bodies. These normally punctate structures are reorganized by E4 ORF3 into tracks that eventually surround viral replication centers. PML rearrangement is an evolutionarily conserved function of E4 ORF3, yet its cause and functional relevance remain mysteries. The E4 ORF3 protein coimmunoprecipitates with the PML protein, yet E4 ORF3 still forms tracks in cells that lack PML. The PML protein is a member of a larger protein family termed tripartite motif (TRIM) proteins. TRIM proteins contain a tripartite domain structure in proximity to their N termini that consists of a RING finger domain, followed by one or two B box domains and a C-terminal coiled-coil domain (collectively termed the RBCC domain). The order and spacing of these domains are evolutionarily conserved and thought to mediate protein-protein interactions and other functions. We implemented a proteomic approach to isolate cellular proteins that bind to E4 ORF3. We identified a novel interaction between E4 ORF3 and another TRIM family member, transcriptional intermediary factor 1 alpha (TIF1␣). TIF1␣ functions by recruiting coactivators and/or corepressors to modulate transcription. The interaction between E4 ORF3 and TIF1␣ was validated by coimmunoprecipitation and binding of recombinant proteins. Indirect immunofluorescence assays demonstrated that TIF1␣ is reorganized into track structures that contain PML upon E4 ORF3 expression. The RBCC domain of TIF1␣ is sufficient for E4 ORF3-induced rearrangement, and TIF1␣ reorganization is conserved across adenovirus serotypes.

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1γ for Proteasome-Dependent Degradation during Infection

“…It has long been established that early during infection E4orf3 reorganizes PML nuclear bodies to form distinct nuclear track-like structures that contain the cellular proteins PML, SP-100, the MRN complex, and TIF1␣ (19,25,65,78). Of these, only the MRN component, MRE11, is subsequently targeted to cytoplasmic aggresomes for E1B-55K/E4orf6-dependent degradation (2,30,44).…”

“…2A). Since it has previously been established that the TIF1 family member, TIF1␣ is reorganized by E4orf3 to PML-containing nuclear tracks in Adinfected cells (78) we next assessed whether TIF1␥ colocalizes with E4orf3 and PML in Ad-infected cells. Consistent with the notion that TIF1␥ might be recruited along with TIF1␣ to PMLcontaining nuclear tracks, these analyses revealed that TIF1␥ colocalized with PML in both Ad5-and Ad12-infected cells (Fig.…”

“…Other work has established that E4orf3 targets the PML II isoform specifically in order to reorganize PML bodies into nuclear tracks and also recruits E1B-55K to these tracks during infection (38,43). More recent work has determined that the transcriptional intermediary factor 1 (TIF1) family member, TIF1␣, is also recruited in an E4orf3-dependent manner to PML nuclear tracks during infection (78). Although E4orf3s from the divergent Ad serotypes Ad4, Ad5, Ad9, and Ad12 all recruit TIF1␣ to PML tracks, the functional importance of TIF1␣ reorganization during infection remains to be established (78).…”

“…More recent work has determined that the transcriptional intermediary factor 1 (TIF1) family member, TIF1␣, is also recruited in an E4orf3-dependent manner to PML nuclear tracks during infection (78). Although E4orf3s from the divergent Ad serotypes Ad4, Ad5, Ad9, and Ad12 all recruit TIF1␣ to PML tracks, the functional importance of TIF1␣ reorganization during infection remains to be established (78). E4orf3, akin to E1B-55K/E4orf6, also targets DNA damage response and repair pathways during infection in order to prevent viral DNA concatenation and doublestrand break repair (DSBR).…”

DNA Virus Replication Compartments

“…Specifically, PML, Sp100, Daxx, SUMO-1, and TIF1␣ are relocated into these tracks by E4orf3 (163)(164)(165)(166), which might resemble nuclear aggresomes (167). These PML-containing structures are thereafter localized adjacent to viral RCs.…”