Multi-subunit tethering complexes participate in the process of vesicle tethering, the initial interaction between transport vesicles and their acceptor compartments. TRAPPII is a highly conserved tethering complex that functions in the late Golgi and consists of all TRAPPI and three specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle electron microscopy. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer responsible for dimerization. TRAPPII binds Ypt1 and likely uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss implications of the TRAPPII structure for coat interaction and TRAPPII-associated human pathologies.

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.

Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.

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 ...

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery. IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such “antiviral” compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

Once transported to the replication sites, human adenoviruses (HAdVs) need to ensure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly characterized but represent a decisive moment in the establishment of a productive infection. Here, we identify a novel host viral restriction factor, KAP1. This heterochromatin-associated transcription factor regulates the dynamic organization of the host chromatin structure via its ability to influence epigenetic marks and chromatin compaction. In response to DNA damage, KAP1 is phosphorylated and functionally inactive, resulting in chromatin relaxation. We discovered that KAP1 posttranslational modification is dramatically altered during HAdV infection to limit the antiviral capacity of this host restriction factor, which represents an essential step required for efficient viral replication. Conversely, we also observed during infection an HAdV-mediated decrease of KAP1 SUMO moieties, known to promote chromatin decondensation events. Based on our findings, we provide evidence that HAdV induces KAP1 deSUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism. IMPORTANCEHere we describe a novel cellular restriction factor for human adenovirus (HAdV) that sheds light on very early modulation processes in viral infection. We reported that chromatin formation and cellular SWI/SNF chromatin remodeling play key roles in HAdV transcriptional regulation. We observed that the cellular chromatin-associated factor and epigenetic reader SPOC1 represses HAdV infection and gene expression. Here, we illustrate the role of the SPOC1-interacting factor KAP1 during productive HAdV growth. KAP1 binds to the viral E1B-55K protein, promoting its SUMO modification, therefore illustrating a crucial step for efficient viral replication. Simultaneously, KAP1 posttranslational modification is dramatically altered during infection. We observed an HAdV-mediated decrease in KAP1 SUMOylation, known to promote chromatin decondensation events. These findings indicate that HAdV induces the loss of KAP1 SUMOylation to minimize epigenetic gene silencing and to promote the SUMO modification of E1B-55K by a so far unknown mechanism.

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.

Human adenoviruses (HAdV) are used as a model system to investigate tumorigenic processes in mammalian cells where the viral oncoproteins E1A and E1B-55K are absolutely required for oncogenic transformation, because they simultaneously accelerate cell cycle progression and inhibit tumor suppressor proteins such as p53, although the underlying mechanism is still not understood in detail. In our present study, we provide evidence that E1B-55K binding to the PML-NB component Sp100A apparently has an essential role in regulating adenovirus-mediated transformation processes. Specifically, when this E1B-55K/Sp100A complex recruits p53, Sp100A-induced activation of p53 transcriptional activity is effectively abolished. Hence, Sp100A exhibits tumor-suppressive activity, not only by stabilizing p53 transactivation but also by depressing E1A/E1B-55K-mediated transformation. E1B-55K counteracts this suppressive activity, inducing Sp100A SUMOylation and sequestering the modified cellular factor into the insoluble matrix of the nucleus or into cytoplasmic inclusions. These observations provide novel insights into how E1B-55K modulates cellular determinants to maintain growth-promoting activity during oncogenic processes and lytic infection.