SummaryAlthough we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin-mediated and dynamin 2-dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src-JNK-AP1 and PI3K-Akt-NF-κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src-JNK signalling to enhance its entry. Activation of these pathways by ultraviolet-inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV-induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3′-dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox-2-catalysed prostaglandin E 2 (PGE2) synthesis and induces expressions of G-protein-coupled E-prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src-JNK-AP1 and PI3K-Akt-NF-κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV-induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox-2-mediated PGE 2/EP receptor signalling pathways, further enhancing Src-JNK-AP1 in a cAMP-dependent manner and PI3K-Akt-NF-κB in a cAMP-independent manner. Accordingly, BEFV stimulates PGE2/EP receptor signalling amplifying Src-JNK-AP1 and PI3K-Akt-NF-κB pathways in an autocrine or paracrine fashion to enhance virus entry.

Autophagy plays an important role in cellular response to pathogens. However, the impact of the autophagy machinery on bovine ephemeral fever virus (BEFV) infection is not yet determined. A recent study in our laboratory demonstrated that BEFV triggers simultaneously the PI3K/Akt/NF-κB and Src/JNK-AP1 pathways in the stage of virus binding to enhance virus entry. In this work, we report that BEFV induces autophagy via upregulation of the PI3K/Akt/NF-κB and Src/JNK/AP1 pathways in the early to middle stages of infection and suppresses the PI3K/Akt/mTOR pathway at the late stage of infection. To activate NF-κB, BEFV promotes degradation of IκBα and activates Akt to stimulate NF-κB translocation into the nucleus. Immunoprecipitation assays revealed that BEFV disrupts Beclin 1 and Bcl-2 interaction by JNK-mediated Bcl-2 phosphorylation, thereby activating autophagy. Overexpression of Bcl-2 reversed the BEFV-induced increase in the LC3 II levels. Suppression of autophagy either by knockdown of autophagy-related genes with shRNAs or treatment with a pharmacological inhibitor 3-MA reduced BEFV replication, suggesting that BEFV-induced autophagy benefits virus replication. Our results revealed that the BEFV M protein is one of the viral proteins involved in inducing autophagy via suppression of the PI3K/Akt/mTORC1 pathway. Furthermore, degradation of p62 was observed by immunoblotting, suggesting that BEFV infection triggers a complete autophagic response. Disruption of autophagosome-lysosome fusion by depleting LAMP2 resulted in reduction of virus yield, suggesting that formation of autolysosome benefits virus production.

Erratum: "Copper electroplating for future ultralarge scale integration interconnection" [J.

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The genomic DNA segment encoding the rpoE gene and its flanking region was cloned from Xanthomonas campestris pv. campestris strain 11 (Xc11). The transcriptional start site of rpoE was located at nucleotide G, which is 33 nucleotides preceding the putative translation initiation codon of rpoE, and a extracytoplasmic function sigma factors (sigma(E))-dependent promoter was identified with -35 (5'-GAACTT-3') and -10 (5'-TCTCA-3') consensus sequences. The protein encoded by rpoE gene acted as a sigma (sigma) factor and was sufficient to direct core RNA polymerase to the rpoE promoter and to stimulate initiation of transcription in vitro. The specific binding of the reconstituted Esigma(E) holoenzyme with the Xc11 rpoE promoter was demonstrated by gel retardation assay and DNAse I footprint analysis. This study clearly demonstrated that the rpoE-rseA-mucD genomic organization of X. campestris is similar to that found in Xylella fastidiosa; however, expression of rpoE in X. campestris is autoregulated by its own sigma(E)-dependent promoter.

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Assemblies of four three-dimensional (3D) mixed-ligand coordination polymers (CPs) having formulas, {[Zn 2 (bdc) 2 (4-bpdh)]·C 2 H 5 OH·2H 2 O} n (1), [Zn(bdc)(4-bpdh)] n (2), {[Zn 2 (bdc) 2 (4-bpdh) 2 ]·(4-bpdh)} n (3), and {[Zn(bdc)(4-bpdh)]·C 2 H 5 OH} n (4) (bdc 2− = dianion of 1,4-benzenedicarboxylic acid, 4-bpdh = 2,5-bis(4-pyridyl)-3,4-diaza-2,4-hexadiene) have been synthesized and structurally characterized by single-crystal X-ray diffraction method. Structural determination reveals that the coordination numbers (geometry) of Zn(II) ions in 1, 2, 3, and 4 are five (distorted square-pyramidal (SP)), six (distorted octahedral (O h )), five (trigonal-bipyramidal (TBP)), and four (tetrahedral (T d )), respectively, and are bridged by 4-bpdh with bis-monodentate coordination mode and bdc 2− ligands with bis-bidentate in 1, chelating/bidentate in 2, bis-monodentate and bis-bidentate in 3, and bis-monodentate in 4, to generate two-fold interpenetrating 3D cube-like metal-organic framework (MOF) with pcu topology, non-interpenetrating 3D MOF, two-fold interpenetrating 3D rectangular-box-like MOF with pcu topology and five-fold interpenetrating diamondoid-like MOF with dia topology, respectively. These different intriguing architectures indicate that the coordination numbers and geometries of Zn(II) ions, coordination modes of bdc 2− ligand, and guest molecules play important roles in the construction of MOFs and the formation of the structural topologies and interpenetrations. Thermal stabilities, and photoluminescence study of 1-4 were also studied in detail. The complexes exhibit ligands based photoluminescence properties at room temperature.

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