Simone Callegari

A deneddylase encoded by Epstein–Barr virus promotes viral DNA replication by regulating the activity of cullin-RING ligases

The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein–Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.

Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells

The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

The Epstein–Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate–dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

Transcription Profiling of Epstein-Barr Virus Nuclear Antigen (EBNA)-1 Expressing Cells Suggests Targeting of Chromatin Remodeling Complexes

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA)-1 regulates virus replication and transcription, and participates in the remodeling of the cellular environment that accompanies EBV induced B-cell immortalization and malignant transformation. The putative cellular targets of these effects of EBNA-1 are largely unknown. To address this issue we have profiled the transcriptional changes induced by short- and long-term expression of EBNA-1 in the EBV negative B-cell lymphoma BJAB. Three hundred and nineteen cellular genes were regulated in a conditional transfectant shortly after EBNA-1 induction while a ten fold higher number of genes was regulated upon continuous EBNA-1 expression. Promoter analysis of the differentially regulated genes demonstrated a significant enrichment of putative EBNA-1 binding sites suggesting that EBNA-1 may directly influence the transcription of a subset of genes. Gene ontology analysis of forty seven genes that were consistently regulated independently on the time of EBNA-1 expression revealed an unexpected enrichment of genes involved in the maintenance of chromatin architecture. The interaction network of the affected gene products suggests that EBNA-1 may promote a broad rearrangement of the cellular transcription landscape by altering the expression of key components of chromatin remodeling complexes.

Herpes virus deneddylases interrupt the cullin-RING ligase neddylation cycle by inhibiting the binding of CAND1

The conserved N-terminal domains of the major tegument proteins of herpes viridae encode cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activity. Here we show that the Epstein-Barr virus-encoded member of this enzyme family, BPLF1, is targeted to cullin-RING ubiquitin ligases (CRLs) via the interaction of the conserved helix-2 with helix-23 of the C-terminal domain (CTD) of cullins, at a site involved in electrostatic interaction with helix-8 of the CRL regulator CAND1. Mutation of the solvent-exposed Asp86 and Asp90 of helix-2 to Arg does not affect the enzymatic activity of BPLF1 but abolishes cullin binding and prevents CRL inactivation. The binding of the catalytically active BPLF1 to cullins inhibits the recruitment of CAND1 to the deneddylated CRLs and promotes the selective degradation of cullins by the proteasome. Cullin proteolysis is rescued by the overexpression of CAND1 or its CTD-binding N-terminal domain. These findings illustrate a new strategy for viral modulation of CRL activity where the combined effects of cullin deneddylation and their targeting for proteasomal degradation drive stable inactivation of the ligases.

Epstein–Barr virus encoded microRNAs target SUMO‐regulated cellular functions

Post‐translational modification by the small ubiquitin‐like modifier (SUMO) regulates the cellular response to different types of stress and plays a pivotal role in the control of oncogenic viral infections. Here we investigated the capacity of microRNAs (miRNAs) encoded by Epstein–Barr virus to interfere with the SUMO signaling network. Using a computational strategy that scores different properties of miRNA–mRNA target pairs, we identified a minimal set of 575 members of the SUMO interactome that may be targeted by one or more Epstein–Barr virus miRNAs. A significant proportion of the candidates cluster in a functional network that controls chromatin organization, stress, DNA damage and immune responses, apoptosis and transforming growth factor beta signaling. Multiple components of the transforming growth factor beta signaling pathway were inhibited upon upregulation of the BamHI‐H rightward open reading frame 1 (BHRF1) encoded miRNAs in cells transduced with recombinant lentiviruses or entering the productive virus cycle. These findings point to the capacity of viral miRNAs to interfere with SUMO‐regulated cellular functions that control key aspects of viral replication and pathogenesis.

An N-terminal SIAH-interacting motif regulates the stability of the ubiquitin specific protease (USP)-19

The Ubiquitin Specific Protease-19 (USP19) regulates cell cycle progression and is involved in the cellular response to different types of stress, including the unfolded protein response (UPR), hypoxia and muscle atrophy. Using the unique N-terminal domain as bait in a yeast-two hybrid screen we have identified the ubiquitin ligases Seven In Absentia Homolog (SIAH)-1 and SIAH2 as binding partners of USP19. The interaction is mediated by a SIAH-consensus binding motif and promotes USP19 ubiquitylation and proteasome-dependent degradation. These findings identify USP19 as a common substrate of the SIAH ubiquitin ligases.

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.

Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.