Adenovirus type 5 E4 Orf3 protein targets promyelocytic leukaemia (PML) protein nuclear domains for disruption via a sequence in PML isoform II that is predicted as a protein interaction site by bioinformatic analysis

Human adenovirus type 5 infection causes the disruption of structures in the cell nucleus termed promyelocytic leukaemia (PML) protein nuclear domains or ND10, which contain the PML protein as a critical component. This disruption is achieved through the action of the viral E4 Orf3 protein, which forms track-like nuclear structures that associate with the PML protein. This association is mediated by a direct interaction of Orf3 with a specific PML isoform, PMLII. We show here that the Orf3 interaction properties of PMLII are conferred by a 40 aa residue segment of the unique C-terminal domain of the protein. This segment was sufficient to confer interaction on a heterologous protein. The analysis was informed by prior application of a bioinformatic tool for the prediction of potential protein interaction sites within unstructured protein sequences (predictors of naturally disordered region analysis; PONDR). This tool predicted three potential molecular recognition elements (MoRE) within the C-terminal domain of PMLII, one of which was found to form the core of the Orf3 interaction site, thus demonstrating the utility of this approach. The sequence of the mapped Orf3-binding site on PML protein was found to be relatively poorly conserved across other species; however, the overall organization of MoREs within unstructured sequence was retained, suggesting the potential for conservation of functional interactions

The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines

Background Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells. Methods Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively. Results AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15. Conclusion Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl

SUMO Pathway Dependent Recruitment of Cellular Repressors to Herpes Simplex Virus Type 1 Genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway

Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling

Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins

Characterisation of Genome-Wide PLZF/RARA Target Genes

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear

A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection

Small ubiquitin-related modifiers: a novel and independent class of autoantigens in primary biliary cirrhosis

Serum autoantibodies against components of nuclear dots (anti-NDs), namely PML and Sp100, are specifically detected in 20% to 30% of patients with primary biliary cirrhosis (PBC). Although anti-ND antibodies are nonpathogenic, the mechanisms that lead to this unique reactivity are critical to understanding the loss of immune tolerance in PBC. Importantly, Sp100 and PML are both covalently linked to small ubiquitin-related modifiers (SUMOs). Therefore, we investigated whether SUMO proteins are independent autoantigens in PBC and studied 99 PBC sera samples for reactivity against NDs, PML, and Sp100, as well as against SUMO-2 and SUMO-1 recombinant proteins. Autoantibodies against SUMO-2 and SUMO-1 were found in 42% and 15% of anti-ND-positive PBC sera, respectively. Anti-SUMO reactivity was not observed in anti-ND-negative sera. Anti-SUMO-2 autoantibodies were found in 58% of sera containing autoantibodies against both PML and Sp100 and were detected exclusively in sera containing anti-Sp100 autoantibodies. In conclusion, SUMO proteins constitute a novel and independent class of autoantigens in PBC. Furthermore, we believe our data emphasize the post-translational modification of lysine by either lipoylation in the case of AMA or SUMOylation in the case of specific anti-ND autoantibodies as the pivotal site for autoantibody generation in PBC