Cross-talk between phosphorylation and SUMOylation regulates transforming activities of an adenoviral oncoprotein

PML nuclear bodies (PML NBs), also called ND10, are matrix-bound nuclear structures that have been implicated in a variety of functions, including DNA repair, transcriptional regulation, protein degradation, and tumor suppression. These domains are also known for their potential to mediate an intracellular defense mechanism against many virus types. This is likely why they are targeted and subsequently manipulated by numerous viral proteins. Paradoxically, the genomes of various DNA viruses become associated with PML NBs, and initial sites of viral transcription/replication centers are often juxtaposed to these domains. The question is why viruses start their transcription and replication next to their supposed antagonists. Here, we report that PML NBs are targeted by the adenoviral (Ad) transactivator protein E1A-13S. Alternatively spliced E1A isoforms (E1A-12S and E1A-13S) are the first proteins expressed upon Ad infection. E1A-13S is essential for activating viral transcription in the early phase of infection. Coimmunoprecipitation assays showed that E1A-13S preferentially interacts with only one (PML-II) of at least six nuclear human PML isoforms. Deletion mapping located the interaction site within E1A conserved region 3 (CR3), which was previously described as the transcription factor binding region of E1A-13S. Indeed, cooperation with PML-II enhanced E1A-mediated transcriptional activation, while deleting the SUMO-interacting motif (SIM) of PML proved even more effective. Our results suggest that in contrast to PML NB-associated antiviral defense, PML-II may help transactivate viral gene expression and therefore play a novel role in activating Ad transcription during the early viral life cycle. Human adenovirus type 5 (Ad5) early region 1A (E1A) is the first protein expressed upon infection and plays an essential role in transcriptional activation and induction of cell cycle progression (1). At early times of infection, two major E1A proteins, E1A-12S (243R) and E1A-13S (289R), are synthesized from alternatively spliced mRNA transcripts of the E1A gene (2, 3). The proteins are identical except for a 46-amino-acid (aa) conserved region (CR3) that is unique to the larger E1A protein (3). Despite being very similar, these proteins show significant differences in their biological activities. The larger E1A isoform (E1A-13S) is considered to be primarily responsible for transactivating viral gene expression by interacting with a wide range of transcription factors via its CR3 (4-8).The PML (promyelocytic leukemia) protein was first described in acute promyelocytic leukemia (APL), showing that PML was fused to retinoic acid receptor alpha (RAR␣) due to a chromosomal translocation (t15;17) (9-20). The PML gene encodes multiple isoforms, derived from alternative mRNA splicing, differing only in the C-terminal parts of the proteins (21). PML seems to be responsible for the assembly of nuclear bodies by recruiting other constitutive components such as Daxx (death domain-associated protein), Sp100 (speckled protein ...

Section: Methodsmentioning

confidence: 99%

The Adenoviral Oncogene E1A-13S Interacts with a Specific Isoform of the Tumor Suppressor PML To Enhance Viral Transcription

Berscheminski

Groitl

Dobner

et al. 2013

“…Intriguingly, SUMO1 is detected mainly in the protein shell formed by PML and Sp100, whereas SUMO2/3 chains are also found in the interior of the PML-NB, mediating interactions with proteins transiently localizing to these domains (46). Since SUMO proteins regulate many different processes within the cell (45,47), it is not surprising that viral pathogens take advantage of this posttranslational modification (PTM) to manipulate cellular pathways and maintain the integrity of viral proteins (48)(49)(50).…”

Section: Importancementioning

confidence: 99%

“…Primary antibodies specific for cellular and ectopically expressed proteins included PML rabbit pAb NB100-59787 (Novus Biologicals, Inc.), Sp100 rabbit pAb GH3 (kindly provided by H. Will), mouse MAb Flag-M2 (Sigma-Aldrich, Inc.), ␤-actin mouse MAb AC-15 (Sigma-Aldrich, Inc.), a 6His mouse MAb (Clontech), and a GFP epitope MAb (Abcam). All protein extracts were prepared in RIPA lysis buffer as described recently (69). For immunoprecipitation of YFP-tagged HP1, a GFP-Trap kit (Chromotek) was used according to the manufacturer's protocol.…”

Section: Plasmids and Transient Transfectionsmentioning

confidence: 99%

Sp100 Isoform-Specific Regulation of Human Adenovirus 5 Gene Expression

Berscheminski

Wimmer

Brun

et al. 2014

Promyelocytic leukemia nuclear bodies (PML-NBs) are nuclear structures that accumulate intrinsic host factors to restrict viral infections. To ensure viral replication, these must be limited by expression of viral early regulatory proteins that functionally inhibit PML-NB-associated antiviral effects. To benefit from the activating capabilities of Sp100A and simultaneously limit repression by Sp100B, -C, and -HMG, adenoviruses (Ads) employ several features to selectively and individually target these isoforms. Ads induce relocalization of Sp100B, -C, and -HMG from PML-NBs prior to association with viral replication centers. In contrast, Sp100A is kept at the PML tracks that surround the newly formed viral replication centers as designated sites of active transcription. We concluded that the host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression. Ad-dependent loss of Sp100 SUMOylation is another crucial part of the virus repertoire to counteract intrinsic immunity by circumventing Sp100 association with HP1, therefore limiting chromatin condensation. We provide evidence that Ad selectively counteracts antiviral responses and, at the same time, benefits from PML-NB-associated components which support viral gene expression by actively recruiting them to PML track-like structures. Our findings provide insights into novel strategies for manipulating transcriptional regulation to either inactivate or amplify viral gene expression. IMPORTANCEWe describe an adenoviral evasion strategy that involves isoform-specific and active manipulation of the PML-associated restriction factor Sp100. Recently, we reported that the adenoviral transactivator E1A targets PML-II to efficiently activate viral transcription. In contrast, the PML-associated proteins Daxx and ATRX are inhibited by early viral factors. We show that this concept is more intricate and significant than originally believed, since adenoviruses apparently take advantage of specific PML-NBassociated proteins and simultaneously inhibit antiviral measures to maintain the viral infectious program. Specifically, we observed Ad-induced relocalization of the Sp100 isoforms B, C, and HMG from PML-NBs juxtaposed with viral replication centers. In contrast, Sp100A is retained at Ad-induced PML tracks that surround the newly formed viral replication centers, acting as designated sites of active transcription. The host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression.

“…Both posttranslational modifications are required for efficient repression of p53-mediated transcription, for the nucleocytoplasmic relocalization of p53, and thus for cellular transformation (25). Furthermore, these modifications are linked in that phosphorylation is required for efficient SUMO conjugation to occur (25). The E1B55K/E4orf6 complex readily shuttles between the nucleus and cytoplasm (26)(27)(28).…”

mentioning

confidence: 99%

“…E1B55K is SUMOylated at its SUMO conjugation motif (SCM) (14,20,21) and phosphorylated on the C-terminal region by casein kinase 2 (CK2) (22)(23)(24). Both posttranslational modifications are required for efficient repression of p53-mediated transcription, for the nucleocytoplasmic relocalization of p53, and thus for cellular transformation (25). Furthermore, these modifications are linked in that phosphorylation is required for efficient SUMO conjugation to occur (25).…”

mentioning

confidence: 99%

Aggresome Formation by the Adenoviral Protein E1B55K Is Not Conserved among Adenovirus Species and Is Not Required for Efficient Degradation of Nuclear Substrates

Blanchette¹,

Wimmer²,

Dallaire³

et al. 2013

bMuch of the work on the basic molecular biology of human adenoviruses has been carried out on a very limited number of the more than 60 serotypes, primarily the highly related species C viruses adenovirus type 5 (Ad5) and Ad2 and, to some extent, Ad12 of species A. Until recently, it has been widely assumed that insights obtained with these model viruses were representative of all human adenoviruses. Recent studies on the E3 ubiquitin ligase formed by the viral E1B55K and E4orf6 proteins with a cellular Cullin-based complex indicated that although all species form such a functional complex, significant variations exist in terms of complex composition and the substrates that are degraded. In the present report we conducted a comprehensive analysis of the localization of E1B55K products from representatives of six of the seven adenovirus species in the presence and the absence of the corresponding E4orf6 protein. We found that although in some species E1B55K localized in aggresomes, such was not always the case, suggesting that these structures are not necessary for the efficient degradation of substrates. In addition, differences were evident in the localization of E1B55K, although all forms readily associated with PML. Finally, Ad5 E1B55K was seen to localize in close proximity to Rab11, a marker for the endosomal recycling compartment, and both focused at the microtubule organizing center. These findings suggest that E1B55K from some species may employ the transport system utilized by the membrane recycling pathway to assemble aggresomes and the possibility that this structure might then affect recycling of cell surface components.