Virion Structure and In Vitro Genome Release Mechanism of Dicistrovirus Kashmir Bee Virus

Infections with Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here, we present the structure of the virion of KBV determined to a resolution of 2.8 angstrom. We show that the exposure of KBV to acidic pH induces a reduction in interpentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae. IMPORTANCE The western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the United States, and no cure for the disease is currently available. Here, we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.We gratefully acknowledge the Cryoelectron Microscopy and Tomography core facility of CEITEC supported by MEYS CR (LM2018127) . This research was carried out under the project CEITEC 2020 (LQ1601) , with financial support from the MEYS of the Czech Republic under National Sustainability Program II. This work was supported by IT4I project (CZ.1.05/1.1.00/02.0070) , funded by the European Regional Development Fund and the national budget of the Czech Republic via the RDIOP, as well as the MEYS via the grant (LM2011033) . The research of G.A.M. was supported by the grants CONICET (PIP 20150288) , 247 Agencia Nacional de Promocion Cientifica y Tecnica, Argentina (PICT no. 2015-248 0665, PICT No. 20181545) , and Universidad Nacional de La Plata, Argentina. The research of D.M.A.G. was supported by a Grupos Consolidados grant from the University of the Basque Country, Spain (GIU18/172) . The research leading to these results received funding from the Grant Agency of the Czech Republic grant GX19-25982X to P.P

Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins

Structural and functional basis of mammalian microRNA biogenesis by Dicer

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways

The Eighth Central European Conference "Chemistry towards Biology": snapshot

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting

Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function

Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of βγ-subunits of G protein transducin (Gtβγ). The ability of Pdc to bind to Gtβγ is inhibited through its phosphorylation at S54 and S73 within the N-terminal domain (Pdc-ND) followed by association with the scaffolding protein 14-3-3. However, the molecular basis for the 14-3-3-dependent inhibition of Pdc binding to Gtβγ is unclear. By using small-angle x-ray scattering, high-resolution NMR spectroscopy, and limited proteolysis coupled with mass spectrometry, we show that phosphorylated Pdc and 14-3-3 form a complex in which the Pdc-ND region 45−80, which forms a part of Pdc’s Gtβγ binding surface and contains both phosphorylation sites, is restrained within the central channel of the 14-3-3 dimer, with both 14-3-3 binding motifs simultaneously participating in protein association. The N-terminal part of Pdc-ND is likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20−30, which are also involved in Gtβγ binding, are positioned close to the surface of the 14-3-3 dimer. The C-terminal domain of Pdc is located outside the central channel and its structure is unaffected by the complex formation. These results indicate that the 14-3-3 protein-mediated inhibition of Pdc binding to Gtβγ is based on steric occlusion of Pdc’s Gtβγ binding surface

Structural basis for the different membrane binding properties of the Escherichia coli anaerobic and human mitochondrial β-oxidation trifunctional enzyme complexes

Facultative anaerobic bacteria such as Escherichia coli have two α2β2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the β-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-β as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-β is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-β dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.Peer reviewe

Ultrastructural details of resistance factors of the bacterial spore revealed by in situ cryo-electron tomography

The bacterial spore owes its incredible resistance capacities to various molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography (cryo-ET) on bacteria lamellae generated by cryo-focused ion beam micromachining (cryo-FIBM) provides insights into the ultrastructural organization of Bacillus subtilis sporangia, including that of the DNA and nascent coat layers. Analysis of the reconstructed tomograms reveal that rather early during sporulation, the chromosome in the developing spore (the forespore) adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a complex stack of amorphous or structured layers with distinct electron density, dimensions and organization. We investigated the nature of the nascent coat layers in various mutant strains using cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE. Significance statement Bacterial spores are dormant cells that can resist to multiple stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans, as in the case of probiotics, or a major problem for public health, food safety or biowarfare when it comes to spores of pathogenic bacteria. In this study, we combined state-of-the-art cryo-electron tomography and conventional cellular electron microscopy to provide insights into intermediate stages of spore development. Our data reveal the intracellular reorganization of the chromosome into a toroidal fibrillar structure and the complex assembly of the multi-protein, multilayered extracellular coat, shedding light on the mechanisms by which the spore acquires its incredible resistance capacities