A Structural Basis for the Assembly and Functions of a Viral Polymer that Inactivates Multiple Tumor Suppressors

Ou, Horng D.; Kwiatkowski, Witek; Deerinck, Thomas J.; Noske, Andrew B.; Blain, Katie Y.; Land, Hannah S.; Soria, Conrado; Powers, Colin; May, Andrew P.; Shu, Xiaokun; Tsien, Roger Y.; Fitzpatrick, James A.J.; Long, J. Bradford De; Ellisman, Mark H.; Choe, Senyon; O’Shea, Clodagh C.

doi:10.1016/j.cell.2012.08.035

Cited by 73 publications

(90 citation statements)

References 75 publications

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“…Here we show that E4-ORF3 self-assembly is critical for SUMO E3 ligase activity in vitro, further substantiating that E4-ORF3 function requires higher-order multimerization, which creates dynamic interfaces with ubiquitin-like proteins, substrates, and enzymes (4,5). This study provides the first direct evidence to our knowledge that E4-ORF3 plays a role in cellular enzymatic processes.…”

Section: Discussionmentioning

confidence: 49%

“…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).…”

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confidence: 99%

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

Sohn

Hearing

2016

Proc. Natl. Acad. Sci. U.S.A.

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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....

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Section: Discussionmentioning

confidence: 49%

mentioning

confidence: 99%

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

Sohn

Hearing

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…This idea is supported by the fact that IE1 does oligomerize, thereby potentially providing an enlarged surface (30). In addition to this, the assumption that the interaction with PML is a prerequisite for IE1 action bears similarity to the case for the adenoviral E4-ORF3 protein, which is suggested to inactivate PML via tight binding using a multivalent matrix built by extensive multimerization (55).…”

Section: Discussionmentioning

confidence: 65%

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

Schilling

Scherer

Reuter

et al. 2017

J Virol

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PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML; however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays, demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner, since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As 2 O 3 -mediated hyperSUMOylation of K160, thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil-mediated PML dimerization, we propose that IE1 affects PML autoSUMOylation either by directly abrogating PML E3 ligase function or by preventing access to SUMO sites. Thus, our data suggest a novel mechanism for how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation. IMPORTANCEThe human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by subnuclear structures termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism for how IE1 antagonizes PML NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility of developing small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.KEYWORDS PML nuclear bodies, human cytomegalovirus, immediate early 1, intrinsic immunity, nuclear domain 10, sumoylation P romyelocytic leukemia protein (PML) is known to act as organizer of subnuclear, matrix-associated structures which can be detected as spherical dots of approximately 0.1 to 1.0 m (1). These structures, termed PML nuclear bodies (NBs) or nuclear domain 10 (ND10), are multiprotein complexes consisting of more than 160 proteins.

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“…The group imaged infected cells by using miniSOG to label the adenovirus oncoprotein E4-ORF3 (ref. 6). Serial block-face scanning EM gave them reconstructed, three-dimensional views of infected cells.…”

Section: Fluorescent Bridgesmentioning

confidence: 99%

Two microscopes are better than one

Smith¹

2012

Nature

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T here are many types of microscopy, each providing a unique set of benefits. Light microscopy of cells that express fluorescently labelled molecules, for example, lets scientists observe the movements of specific molecules or protein complexes in live cells in real time, or in fixed samples. Scanning electron microscopy (EM) reveals tiny details of the cell surface, and transmission EM shows the detailed cytoarchitecture of sections through fixed tissue. Other aspects of the structure of cells and tissues can be explored using techniques such as ion microscopy, total internal reflection microscopy, atomic force microscopy and super-resolution microscopy. Each type provides different information, but using two sorts of microscopy simultaneously provides even more -and molecular tools have been developed to link them together. Researchers testing the waters of this 'correlative microscopy' are beginning to discover its challenges and rewards.Correlated light-electron microscopy, for example, provides both the specificity and real-time observation of light microscopy with fluorescent labelling and the better structural resolution of EM. But such correlative microscopy has historically been tricky to use. Scientists who tried it might have needed to switch to EM halfway through an experiment, which meant moving the sample from one This image of a butterfly wing was constructed using two types of microscopy: a confocal image of the reflective eyespots shows scales (green) and wing (red); scanning electron microscopy reveals the different structure of non-reflective scales (upper left).

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A Structural Basis for the Assembly and Functions of a Viral Polymer that Inactivates Multiple Tumor Suppressors

Cited by 73 publications

References 75 publications

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

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

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

Two microscopes are better than one

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