Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

No abstract

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.

bThe role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

No abstract

nature structural & molecular biology advance online publication a r t i c l e sAlthough undisputable evidence has clearly demonstrated that the nuclear steps of mRNA processing are mechanistically linked to transcription, a conceptual evolution in gene regulation has come with the realization that transcription might also be functionally connected to more remote processes occurring in the cytoplasm, such as mRNA decay 1,2 . Cytoplasmic mRNA decay is initiated by deadenylation, a rate-limiting event during which the poly(A) tail of the transcript is trimmed off by the CCR4-NOT complex, the main deadenylation machinery in eukaryotes 3 . The degradation of specific mRNAs, a key process in the regulation of eukaryotic gene expression, is achieved through the recruitment of the CCR4-NOT complex by sequence-specific RNA-binding proteins (RBPs) or by the microRNA machinery 3,4 . Poly(A)-shortened mRNAs, along with factors involved in the deadenylation, decapping and mRNA-degradation machineries, accumulate in microscopic mRNA-protein complex (mRNP) aggregates called processing bodies (PBs) 5 .The idea of coupling between mRNA synthesis and degradation has recently emerged. Genome-wide expression studies in yeast have shown that mRNA synthesis and decay are mechanistically and functionally coordinated, thus supporting the existence of common molecular effectors [6][7][8][9] . In particular, the CCR4-NOT deadenylation complex was first described as a transcriptional regulator and has been implicated in initiation and elongation by RNA polymerase II 10,11 . More surprisingly, it has also been shown that degradation of yeast mRNAs is determined by cis-acting sequence elements in promoters 12,13 . These findings have led to the concept of mRNA imprinting, in which sequence-specific DNA-binding factors might orchestrate mRNA synthesis and decay. This decay would occur via loading of factors regulating cytoplasmic mRNA degradation onto the transcribing mRNA 14 . However, the identity of these DNA-binding mRNA coordinators is still obscure, and it remains to be tested whether such coupling between transcription and decay also exists in higher eukaryotes. E26 (Ets) proteins, a family of 28 helix-loop-helix transcription factors (TFs) in metazoans, are characterized by a highly conserved DNA-binding ETS domain 15 . Through this domain, Ets factors bind specific gene promoters and act as key regulators in many biological processes including cellular proliferation, apoptosis, differentiation and survival 16 . ERG, FLI1 and the more structurally divergent FEV compose the Erg subfamily of Ets factors and have been identified as driving factors in prostate cancer, Ewing's tumors and leukemias 15,17 . Using ERG as a paradigm, we sought to investigate the possibility that eukaryotic transcription factors might be directly involved in cytoplasmic mRNA decay. We demonstrate that ERG triggers degradation of mRNAs connected to Aurora signaling by recruiting RBPs and the CCR4-NOT deadenylation complex and that this activity is important fo...