Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 Å, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU''s preference for Ub and Ub-like substrates.Crimean-Congo hemorrhagic fever (CCHF) is characterized in humans by the sudden onset of fever, myalgia, headache, dizziness, sore eyes, photophobia, and hyperanemia as well as severe hemorrhages (28, 43, 46). The causative agent of CCHF is the CCHF virus, which is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] virus of the genus Nairovirus, belonging to the viral family Bunyaviridae. Originally named after outbreaks in the former Soviet Union and in the Congo during the mid-20th century, the affected area of this disease has rapidly spread to large areas of sub-Saharan Africa, the Balkans, Northern Greece, European Russia, Pakistan, the Arabian Peninsula, Iran, Afghanistan, Iraq, Turkey, and recently, the Xinjiang province of China (43, 46). The CCHF viral genome, as well as those of the closely related Dugbe and Nairobi viruses, consists of three negative-sense RNA segments, small (S), medium (M), and large (L). Incubation of CCHF is 5 to 6 days, with fatalities occurring less than 7 days after signs of infection. Fatality rates for patients infected with the CCHF virus ranged from 5% to 70%, depending on phylogenetic variation of the virus, transmission route, treatment facility, and the reporting and confirmation of the case statistics (19, 32, 43, 47).The innate immune system serves as the human''s first line of defense from invading pathogens, including CCHF virus. The type I interferon (IFN) response comprises a key component of this system by upregulating more than 300 IFN-stimulated genes (ISGs) whose products detect viral molecules, promote amplification of the type I IFN response, modulate other signaling pathways, and directly provide antiviral activity (34). Regulation of the type I IFN response has been shown to rely on posttranslational modification by ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15) (14, 23). Both Ub and ISG15 are expressed in a proform and cleaved to leave a double-glycine C terminus that forms an isopeptide bond with predominantly the ɛ-NH₂ of lysine residues of a target protein through a three-step enzymatic process. In addition to forming isopeptide bonds with target proteins, Ub, which contains seven lysine residues, has been observed to form poly-Ub chains. The most studied of these moieties are K29-linked, K48-linked, and K63-linked poly-Ub. While K29-linked and K48-linked polyubiquitination of proteins leads to their degradation in the lysosome and proteasome, respectively, conjugation of K63-linked poly-Ub to proteins has an activating effect, resulting in an enhanced type I IFN response (2, 7, 18, 33, 40). Currently, more than 150 proteins have been identified as forming conjugates with ISG15, with the number of proteins forming Ub conjugates far exceeding that number (12, 48). A subset of type I IFN signaling and effector proteins that Ub and ISG15 have been shown to stabilize includes JAK1, STAT1/2, double-stranded RNA-dependent protein kinase (PKR), myxovirus-resistant protein A (MxA), and RIG-I (17). MxA has particularly shown to be important in type I IFN response to CCHF infection. RIG-I and several other proteins have also been shown to be targets for K63-linked poly-Ub (4).Recently, investigators have identified a cysteine viral ovarian tumor domain (vOTU) protease colocated with the RNA-dependent RNA polymerase in the L protein of the CCHF virus (14). Interestingly, as CCHF is an ssRNA(−) virus, no protease is required to cleave a viral polypeptide to facilitate viral replication as in positive-sense ssRNA [ssRNA(+)] viruses. Furthermore, recent reports have observed that vOTU is not required for RNA-dependent RNA polymerase activity and for vOTU protease activity linked to impairment of the type I IFN response through its deubiquitinating and deISGylating activity (6, 14). Additional studies have also tentatively identified the presence of vOTU homologues in the Arterivirus genus of the Arteriviridae family, suggesting that they too may facilitate impairment of the type I IFN response (14). Since the discovery of the first ovarian tumor domain (OTU) protease in Drosophila oogenesis and prior to the identification of vOTU, OTU superfamily members could be divided into three subclasses according to their sequence homology, otubains, A20-like OTUs, and ubiquitin thioesterase ZRANB1 (22). With the addition of the viral OTU subclass, OTU superfamily members in more than 100 eukaryotic, bacterial, and viral proteins have now been identified (6, 27). Predominantly, OTU proteases have been linked to ubiquitin (Ub) removal and/or remodeling of Ub-conjugated proteins, placing them among five protease superfamilies that facilitate signal transduction cascades and play key roles in protein stability (22). However, vOTU is unique in that it is the only OTU to have shown both deubiquitinating and deISGylating activity (14). Instead, Otubain1/2 (OTUB1/2) plays a key role in T cell response and prefers K48-linked poly-Ub or NEDD8 (neural precursor cell expressed, developmentally downregulated 8) as a substrate (12). A20 and A20-like Cezanne OTU proteases are negative regulators of the NF-κB-mediated inflammation response, selectively cleaving K63-linked poly-Ub targets. DUBA also shows preference for K63-linked poly-Ub (20). In attempts to better understand the OTU superfamily, structures of OTUB and A20-like OTU domains have been elucidated (12, 21, 30). An X-ray structure of the yeast ovarian tumor 1 (yOTU1) domain, which interacts with Cdc48 and has a preference for K48-linked poly-Ub, was achieved in complex with mono-Ub (27). However, since yOTU1 has a preference for K48-linked Ub and possesses low sequence identity to vOTU and other OTU domain proteases, only limited information on vOTU could be obtained. In addition to vOTU, several other viral proteases, such as papain-like protease (PLpro) from the severe acute respiratory syndrome (SARS) coronavirus, have also shown deubiquitinating and deISGylating activity to evade the innate immune system (6, 8, 43, 49). However, no viral proteases that are known to possess deISGylating activity have been visualized as being bound to Ub or Ub-like substrates. To address this issue and elucidate the atomic-level structure of a member from the viral OTU superfamily subclass, we have obtained the X-ray crystal structure of vOTU bound with Ub (vOTU-Ub). We also have characterized the vOTU substrate specificity for mono-Ub, ISG15, and NEDD8 and compared the results with those from human OTUB2 (hOTUB2). Additionally, we assessed vOTU''s deubiquitinating activity toward K48- and K63-linked poly-Ub.     

Interferon regulatory factor 4 (IRF-4) targets IRF-5 to regulate Epstein-Barr virus transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdown-mediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation.     

Receptor-isoform-selective insulin analogues give tissue-preferential effects

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood-glucose-lowering effect upon acute intravenous dosing of mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR-isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on IR-A/IR-B expression.     

Low genetic differentiation among reed warbler Acrocephalus scirpaceus populations across Europe

Migratory birds generally have higher dispersal propensity than resident species and are thus expected to show less genetic differentiation. On the other hand, specific migration patterns may promote genetic structure, such as in situations where migratory divides impede random mixing of individuals. Here we investigated population genetic structure and gene flow patterns in a polytypic passerine, the reed warbler Acrocephalus scirpaceus which shows a migratory divide in central Europe. Using ten polymorphic microsatellite loci and extensive sampling we found low but significant overall genetic differentiation (F_ST=0.013, G’_ST=0.078, D=0.063). Hierarchical F‐statistics and barrier analyses showed low but significant genetic differentiation of Iberian populations, and also slight genetic differences across the migratory divide and between subspecies (A. s. scirpaceus and A. s. fuscus). Three individual‐based Bayesian methods, however, inferred a single genetic unit. Our study thus found low levels of genetic differentiation among reed warbler populations but this genetic differentiation was not pronounced enough to detect a clear population structure using the microsatellite data and no prior information on geographic location of the sampled individuals. This result indicates high levels of gene flow and suggests a possibly recent divergence of European populations after a rapid range expansion. Further studies are necessary to assess divergence times and to reveal the evolutionary history of the reed warbler populations.     

The role of canonical Wnt signaling in leg regeneration and metamorphosis in the red flour beetle Tribolium castaneum

Many organisms across the Metazoa have regenerative abilities with potentially conserved genetic mechanisms that can enlighten both medicine and evolutionary studies. Here, the role of canonical Wnt signaling was examined in the red flour beetle Tribolium castaneum in order to explore its role during metamorphosis and larval leg regeneration. Double-stranded RNA mediated silencing of Wnt-1 signaling resulted in a loss of wings and appendages with a dramatic reduction in width, indicating that the Wnt-1 signaling pathway is necessary for proper post-embryonic appendage development in T. castaneum. Furthermore, disruption of canonical Wnt signaling led to the complete impairment of limb regeneration in T. castaneum. Our findings suggest that Wnt-1 signaling is a conserved mechanism for appendage development across all holometabolous insects and indicate that the role of Wnt-1 signaling in limb regeneration has been retained across all insects as various modes of limb development evolved. Importantly, this study shows that the availability of the genome sequence and the ease of performing leg ablations make Tribolium an excellent holometabolous insect model for studying regeneration.     

Unique arbuscular mycorrhizal fungal communities uncovered in date palm plantations and surrounding desert habitats of Southern Arabia

The main objective of this study was to shed light on the previously unknown arbuscular mycorrhizal fungal (AMF) communities in Southern Arabia. We explored AMF communities in two date palm (Phoenix dactylifera) plantations and the natural vegetation of their surrounding arid habitats. The plantations were managed traditionally in an oasis and according to conventional guidelines at an experimental station. Based on spore morphotyping, the AMF communities under the date palms appeared to be quite diverse at both plantations and more similar to each other than to the communities under the ruderal plant, Polygala erioptera, growing at the experimental station on the dry strip between the palm trees, and to the communities uncovered under the native vegetation (Zygophyllum hamiense, Salvadora persica, Prosopis cineraria, inter-plant area) of adjacent undisturbed arid habitat. AMF spore abundance and species richness were higher under date palms than under the ruderal and native plants. Sampling in a remote sand dune area under Heliotropium kotschyi yielded only two AMF morphospecies and only after trap culturing. Overall, 25 AMF morphospecies were detected encompassing all study habitats. Eighteen belonged to the genus Glomus including four undescribed species. Glomus sinuosum, a species typically found in undisturbed habitats, was the most frequently occurring morphospecies under the date palms. Using molecular tools, it was also found as a phylogenetic taxon associated with date palm roots. These roots were associated with nine phylogenetic taxa, among them eight from Glomus group A, but the majority could not be assigned to known morphospecies or to environmental sequences in public databases. Some phylogenetic taxa seemed to be site specific. Despite the use of group-specific primers and efficient trapping systems with a bait plant consortium, surprisingly, two of the globally most frequently found species, Glomus intraradices and Glomus mosseae, were not detected neither as phylogenetic taxa in the date palm roots nor as spores under the date palms, the intermediate ruderal plant, or the surrounding natural vegetation. The results highlight the uniqueness of AMF communities inhabiting these diverse habitats exposed to the harsh climatic conditions of Southern Arabia.     

RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

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Highlights? A common set of target genes directly regulated by MLL-AF4 is identified ? RUNX1 is a target gene that is specifically upregulated in t(4;11) patients ? MLL-AF4 controls RUNX1 gene expression by stabilizing ENL and AF9 binding ? RUNX1 cooperates with an AF4-MLL complex to activate gene targets