Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule’s necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.DOI: http://dx.doi.org/10.7554/eLife.25940.001

Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (PCs). The PCs are required for the oral toxicity of BoNT in the context of food-borne botulism and are thought to protect BoNT from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. The PC can differ in size and protein content depending on the C. botulinum strain. The oral toxicity of the BoNT PC increases as the size of the PC increases, but the molecular architecture of these large complexes and how they contribute to BoNT toxicity have not been elucidated. We have generated 2D images of PCs from strains producing BoNT serotypes A1, B, and E using negative stain electron microscopy and single-particle averaging. The BoNT/A1 and BoNT/B PCs were observed as ovoid-shaped bodies with three appendages, whereas the BoNT/E PC was observed as an ovoid body. Both the BoNT/A1 and BoNT/B PCs showed significant flexibility, and the BoNT/B PC was documented as a heterogeneous population of assembly/disassembly intermediates. We have also determined 3D structures for each serotype using the random conical tilt approach. Crystal structures of the individual proteins were placed into the BoNT/A1 and BoNT/B PC electron density maps to generate unique detailed models of the BoNT PCs. The structures highlight an effective platform that can be engineered for the development of mucosal vaccines and the intestinal absorption of oral biologics.hemagglutinin | nontoxic nonhemagglutinin | sialic acid

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of Synaptotagmin (Syt)-I and SytII, homologous synaptic vesicle proteins. We observe conditions in which BoNT/B binds both Syt isoforms, but BoNT/G only binds SytI. Both serotypes bind ganglioside G T1b . The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform to understand the physiological relevance of different Syt receptor isoforms in vivo.Botulinum neurotoxin (BoNT) is the causative agent of botulism, a potentially lethal neuroparalytic condition in humans (1). The extreme potency of BoNT (LD 50 value ∼ 0.1 ng kg -1 ) stems from the toxin's high affinity for neuronal receptors at the neuromuscular junction and enzymatic inhibition of neurotransmitter release (2). BoNT's are produced as single chain proteins in seven antigenically distinct forms (serotypes A-G). Most BoNT serotypes undergo post-translational cleavage to form a dichain molecule composed of a light-and heavy-chain linked by a disulfide bond. The light chain (LC) is a zinc metalloprotease that cleaves SNARE proteins to inhibit neurotransmitter vesicle fusion to the plasma membrane (3). The N-terminal half of the heavy chain (HCT) is involved in translocation of the LC across the endosomal membrane, and the C-terminal half of the heavy chain (HCR) is involved in binding receptors (4).BoNT targets the neuromuscular junction through specific interactions with both ganglioside and protein receptors (5). BoNTs bind G D1a and gangliosides in the G 1b series and show the highest affinity for the trisialoganglioside, G T1b (6). The protein receptor can vary with BoNT serotype. Synaptotagmin (Syt)-I and SytII mediate the internalization of BoNT/B and/G, but not BoNT/A or/E, into neuronal cells (7-9). SytI and SytII are homologous calcium sensors that couple neuronal calcium influx to the fast phase of neurotransmitter release (10). BoNT/B and/G bind to the luminal domains of SytI and SytII following the fusion of synaptic vesicles with the plasma membrane. The ability of a peptide, corresponding to 20 amino acids of the SytII luminal domain, in conjunction with gangliosides, to neutralize BoNT/B toxicity in mice is consistent with the SytII luminal domain being the neuronal receptor for BoNT/B (7).* To whom correspondence should be addressed. jtb01@mcw.edu; phone (414) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBoNT/G is a recently discovered serotype whose HCR shares a high degree of primary amino acid conservation with BoNT/B (50% identical, 71% similar). Despite the homology between BoNT/B and BoNT/G and between SytI and SytII, several differences exist in their interactions with neurons. BoNT/B binds SytII independent of ganglioside but requires gangliosid...

We report the synthesis and full characterization for a series of thiacrown complexes of Pt(II) incorporating the fluxional trithiacrown ligand 1,4,7-trithiacyclononane ([9]aneS3) and several group 15 donors ligands. Reaction of [Pt([9]aneS3)Cl2] with a full stoichiometric equivalent of the group 15 donor (L = 2 x AsPh3, SbPh3 or 1,2-bis(diphenylarsenio) ethane (dpae) followed by metathesis with NH4PF6 yields [Pt([9]aneS3)L](PF6)2. We also report the analogous Pd(II) complex with dpae. Similar reactions of the starting Pt complex with one equivalent of XPh3 (X = As or Sb) result in complexes of the formula [Pt([9]aneS3)(XPh3)(Cl)](PF6). All six new complexes have been fully characterized by multinuclear NMR, IR, and UV-Vis spectroscopies in addition to elemental analysis and single crystal structural determinations. The X-ray structures of each complex indicate an axial M-S interaction formed by the endodentate conformation of the [9]aneS3 ligand. The axial M-S distance is highly dependent upon the ancillary donor set. The axial M-S distance shortens with the identity of the group 15 donor ligand according to the trend, Sb < As < P, due to their increasingly poorer donor qualities. The two bis pnictogen complexes, [Pt([9]aneS3)(AsPh3)2](PF6)2 and [Pt([9]aneS3)(SbPh3)2](PF6)2 form unusual five-coordinate distorted trigonal bipyramids in contrast to the pseudo-five coordinate, elongated square pyramidal structures typically observed in Pt(II) complexes of [9]aneS3. The distortion arises from intramolecular pi-pi interactions between the phenyl rings on the two different triphenyl ligands. Chemical shifts in the 195Pt NMR also show similar periodic relationships which trend progressively upfield as the donor atom becomes larger. As expected, the coordinated [9]aneS3 ligand shows fluxional behavior in its NMR spectra, resulting in a single 13C NMR resonance, despite the asymmetric coordination environment found in both chloro complexes. The line width for the carbon NMR resonance as well as for the 195Pt NMR peak is highly sensitive to the nature of the group 15 donor, with poorer donors such as SbPh3 showing significant line broadening. Measurements from the electronic spectra support that the ligand field strength of the pnictogen donor decreases with its increasing size.