SUMOylation modulates the stability and function of PI3K-p110ß.

Class I PI3K are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit involved in multiple cellular functions. Recently, the catalytic subunit p110ß has emerged as a class I PI3K isoform playing a major role in tumorigenesis. Understanding its regulation is crucial for the control of the PI3K pathway in p110ß-driven cancers. Here we sought to evaluate the putative regulation of p110ß by SUMO. Our data show that p110ß can be modified by SUMO1 and SUMO2 in vitro, in transfected cells and under completely endogenous conditions, supporting the physiological relevance of p110ß SUMOylation. We identify lysine residue 952, located at the activation loop of p110ß, as essential for SUMOylation. SUMOylation of p110ß stabilizes the protein increasing its activation of AKT which promotes cell growth and oncogenic transformation. Finally, we show that the regulatory subunit p85ß counteracts the conjugation of SUMO to p110ß. In summary, our data reveal that SUMO is a novel p110ß interacting partner with a positive effect on the activation of the PI3K pathway.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function.

The ribosomal protein L11 (RPL11) integrates different types of stress into a p53-mediated response. Here, we analyzed the impact of the ubiquitin-like protein SUMO on the RPL11-mouse double-minute 2 homolog-p53 signaling. We show that small ubiquitin-related modifier (SUMO)1 and SUMO2 covalently modify RPL11. We find that SUMO negatively modulates the conjugation of the ubiquitin-like protein neural precursor cell-expressed developmentally downregulated 8 (NEDD8) to RPL11 and promotes the translocation of the RP outside of the nucleoli. Moreover, the SUMO-conjugating enzyme, Ubc9, is required for RPL11-mediated activation of p53. SUMOylation of RPL11 is triggered by ribosomal stress, as well as by alternate reading frame protein upregulation. Collectively, our data identify SUMO protein conjugation to RPL11 as a new regulator of the p53-mediated cellular response to different types of stress and reveal a previously unknown SUMO-NEDD8 interplay.-El Motiam, A., Vidal, S., de la Cruz-Herrera, C. F., Da Silva-Álvarez, S., Baz-Martínez, M., Seoane, R., Vidal, A., Rodríguez, M. S., Xirodimas, D. P., Carvalho, A. S., Beck, H. C., Matthiesen, R., Collado, M., Rivas, C. Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function.

Activation of the double-stranded RNA-dependent protein kinase PKR by small ubiquitin-like modifier (SUMO).

The dsRNA-dependent kinase PKR is an interferon-inducible protein with ability to phosphorylate the α subunit of the eukaryotic initiation factor (eIF)-2 complex, resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. Here we analyzed the modification of PKR by the small ubiquitin-like modifiers SUMO1 and SUMO2 and evaluated the consequences of PKR SUMOylation. Our results indicate that PKR is modified by both SUMO1 and SUMO2, in vitro and in vivo. We identified lysine residues Lys-60, Lys-150, and Lys-440 as SUMOylation sites in PKR. We show that SUMO is required for efficient PKR-dsRNA binding, PKR dimerization, and eIF2α phosphorylation. Furthermore, we demonstrate that SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA, whereas a PKR SUMOylation mutant is impaired in its ability to inhibit protein synthesis and shows reduced capability to control vesicular stomatitis virus replication and to induce apoptosis in response to vesicular stomatitis virus infection. In summary, our data demonstrate the important role of SUMO in processes mediated by the activation of PKR.

Expression of the Ebola Virus VP24 Protein Compromises the Integrity of the Nuclear Envelope and Induces a Laminopathy-Like Cellular Phenotype.

Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein that inhibits interferon (IFN) gene expression and counteracts the IFN-mediated antiviral response, preventing nuclear import of signal transducer and activator of transcription 1 (STAT1). Proteomic studies to identify additional EBOV VP24 partners have pointed to the nuclear membrane component emerin as a potential element of the VP24 cellular interactome. Here, we have further studied this interaction and its impact on cell biology. We demonstrate that VP24 interacts with emerin but also with other components of the inner nuclear membrane, such as lamin A/C and lamin B. We also show that VP24 diminishes the interaction between emerin and lamin A/C and compromises the integrity of the nuclear membrane. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, expression of VP24 also promoted the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), a common interactor of lamin A/C and emerin, leading to repression of the BAF-regulated CSF1 gene. Importantly, we found that EBOV infection results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. In summary, here we demonstrate that VP24 acts at the nuclear membrane, causing morphological and functional changes in cells that recapitulate several of the hallmarks of laminopathy diseases. IMPORTANCE The Ebola virus (EBOV) VP24 protein is a nucleocapsid-associated protein with multiple functions. Proteomic studies have identified the cellular nuclear membrane component emerin as a potential VP24 interactor. Here, we demonstrate that VP24 not only interacts with emerin but also with lamin A/C and lamin B, prompting nuclear membrane disruption. This disruption is associated with nuclear morphological abnormalities, activation of a DNA damage response, the phosphorylation of extracellular signal-regulated kinase (ERK), and the induction of interferon-stimulated gene 15 (ISG15). Interestingly, VP24 also promotes the cytoplasmic translocation and downmodulation of barrier-to-autointegration factor (BAF), leading to repression of the BAF-regulated CSF1 gene. Finally, we show that EBOV infection also results in the activation of pathways associated with nuclear envelope damage, consistent with our observations in cells expressing VP24. These results reveal novel activities of EBOV VP24 protein, resulting in a cell phenotype similar to that of most laminopathies, with potential impact on EBOV replication.

Susceptibility of Zebrafish to Vesicular Stomatitis Virus Infection.

The zebrafish, Danio rerio, has become recognized as a valuable model for infectious diseases. Here we evaluated the susceptibility of zebrafish to be infected with the mammalian vesicular stomatitis virus (VSV). Both zebrafish cells and embryos were highly susceptible to VSV infection. Mortalities exceeded 80% in infected embryos and were preceded by the invasion of the central nervous system by VSV. Live imaging of the infection with GFP-VSV as well as virus titration from infected fish confirmed the viral replication. Immunohistochemical analysis of embryonic fish provided evidence of viral antigens as well as of the apoptosis marker caspase-3 in the brain, eye, liver, pronephros, and skeletal muscle. So far, this is the first report describing the susceptibility of zebrafish to the mammalian virus VSV.

Phosphorylable tyrosine residue 162 in the double-stranded RNA-dependent kinase PKR modulates its interaction with SUMO.

Activated dsRNA-dependent serine/threonine kinase PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), resulting in a shut-off of general translation, induction of apoptosis, and inhibition of virus replication. PKR can be activated by binding to dsRNA or cellular proteins such as PACT/RAX, or by its conjugation to ISG15 or SUMO. Here, we demonstrate that PKR also interacts with SUMO in a non-covalent manner. We identify the phosphorylable tyrosine residue 162 in PKR (Y162) as a modulator of the PKR-SUMO non-covalent interaction as well as of the PKR SUMOylation. Finally, we show that the efficient SUMO-mediated eIF2α phosphorylation and inhibition of protein synthesis induced by PKR in response to dsRNA depend on this residue. In summary, our data identify a new mechanism of regulation of PKR activity and reinforce the relevance of both, tyrosine phosphorylation and SUMO interaction in controlling the activity of PKR.

Regulation of Ebola virus VP40 matrix protein by SUMO.

The matrix protein of Ebola virus (EBOV) VP40 regulates viral budding, nucleocapsid recruitment, virus structure and stability, viral genome replication and transcription, and has an intrinsic ability to form virus-like particles. The elucidation of the regulation of VP40 functions is essential to identify mechanisms to inhibit viral replication and spread. Post-translational modifications of proteins with ubiquitin-like family members are common mechanisms for the regulation of host and virus multifunctional proteins. Thus far, no SUMOylation of VP40 has been described. Here we demonstrate that VP40 is modified by SUMO and that SUMO is included into the viral like particles (VLPs). We demonstrate that lysine residue 326 in VP40 is involved in SUMOylation, and by analyzing a mutant in this residue we show that SUMO conjugation regulates the stability of VP40 and the incorporation of SUMO into the VLPs. Our study indicates for the first time, to the best of our knowledge, that EBOV hijacks the cellular SUMOylation system in order to modify its own proteins. Modulation of the VP40-SUMO interaction may represent a novel target for the therapy of Ebola virus infection.

Cell senescence is an antiviral defense mechanism.

Cellular senescence is often considered a protection mechanism triggered by conditions that impose cellular stress. Continuous proliferation, DNA damaging agents or activated oncogenes are well-known activators of cell senescence. Apart from a characteristic stable cell cycle arrest, this response also involves a proinflammatory phenotype known as senescence-associated secretory phenotype (SASP). This, together with the widely known interference with senescence pathways by some oncoviruses, had led to the hypothesis that senescence may also be part of the host cell response to fight virus. Here, we evaluate this hypothesis using vesicular stomatitis virus (VSV) as a model. Our results show that VSV replication is significantly impaired in both primary and tumor senescent cells in comparison with non-senescent cells, and independently of the stimulus used to trigger senescence. Importantly, we also demonstrate a protective effect of senescence against VSV in vivo. Finally, our results identify the SASP as the major contributor to the antiviral defense exerted by cell senescence in vitro, and points to a role activating and recruiting the immune system to clear out the infection. Thus, our study indicates that cell senescence has also a role as a natural antiviral defense mechanism.

KSHV latent protein LANA2 inhibits sumo2 modification of p53.

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.