Purification and electron cryomicroscopy of coronavirus particles.

Intact, enveloped coronavirus particles vary widely in size and contour, and are thus refractory to study by traditional structural means such as X-ray crystallography. Electron microscopy (EM) overcomes some problems associated with particle variability and has been an important tool for investigating coronavirus ultrastructure. However, EM sample preparation requires that the specimen be dried onto a carbon support film before imaging, collapsing internal particle structure in the case of coronaviruses. Moreover, conventional EM achieves image contrast by immersing the specimen briefly in heavy-metal-containing stain, which reveals some features while obscuring others. Electron cryomicroscopy (cryo-EM) instead employs a porous support film, to which the specimen is adsorbed and flash-frozen. Specimens preserved in vitreous ice over holes in the support film can then be imaged without additional staining. Cryo-EM, coupled with single-particle image analysis techniques, makes it possible to examine the size, structure and arrangement of coronavirus structural components in fully hydrated, native virions. Two virus purification procedures are described

Intensity of Brillouin light scattering from spin waves in magnetic multilayers with noncollinear spin configurations: Theory and experiment

The scattering of photons from spin waves (Brillouin light scattering -- BLS) is a well-established technique for the study of layered magnetic systems. The information about the magnetic state and properties of the sample is contained in the frequency position, width, and intensity of the BLS peaks. Previously [Phys. Rev. B 67, 184404 (2003)], we have shown that spin wave frequencies can be conveniently calculated within the ultrathin film approach, treating the intralayer exchange as an effective bilinear interlayer coupling between thin virtual sheets of the ferromagnetic layers. Here we give the consequent extension of this approach to the calculation of the Brillouin light scattering (BLS) peak intensities. Given the very close relation of the BLS cross-section to the magneto-optic Kerr effect (MOKE), the depth-resolved longitudinal and polar MOKE coefficients calculated numerically via the usual magneto-optic formalism can be employed in combination with the spin wave precessional amplitudes to calculate full BLS spectra for a given magnetic system. This approach allows an easy calculation of BLS intensities even for noncollinear spin configurations including the exchange modes. The formalism is applied to a Fe/Cr/Fe/Ag/Fe trilayer system with one antiferromagnetically coupling spacer (Cr). Good agreement with the experimental spectra is found for a wide variety of spin configurations.Comment: 19 pages, 5 figure

Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3.

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1''-phosphate (Appr-1''-p). The SARS nsP3 domain readily removes the 1'' phosphate group from Appr-1''-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1''-p

The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco. Although the enzymes and mechanisms involved in these steps are well characterized, the regulation of eukaryotic Moco biosynthesis is not. Within this work, we described the regulation of Moco biosynthesis in the filamentous fungus Neurospora crassa, which revealed the first step of the multi-step pathway to be under transcriptional control. We found, that upon the induction of high cellular Moco demand a single transcript variant of the nit-7 gene is increasingly formed pointing towards, that essentially the encoded enzyme NIT7-A is the key player for Moco biosynthesis activity in Neurospora

Oligonucleotide compound and method for treating nidovirus infections

A method and oligonucleotide compound for inhibiting replication of a nidovirus in virus-infected animal cells are disclosed. The compound (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the infected cells, (iii) contains between 8-25 nucleotide bases, and (iv) has a sequence capable of disrupting base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region. In practicing the method, infected cells are exposed to the compound in an amount effective to inhibit viral replication

Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes

Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12

Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE–lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription

Contribution of the PhoP/Q regulon to survival and replication of Salmonella enterica serovar Typhimurium in macrophages

The ability of serovars of Salmonella enterica to cause systemic disease is dependent upon their survival and replication within macrophages. To do this, bacteria must withstand or surmount bacteriostatic and bactericidal responses by the host cell, including the delivery of hydrolytic enzymes from lysosomes to the phagosome. The bacterial two-component regulatory system PhoP/Q has been implicated in avoidance of phagolysosomal fusion by S. enterica serovar Typhimurium (S. Typhimurium) in murine macrophages. In this study, the involvement of PhoP/Q-activated genes in avoidance of phagolysosomal fusion was analysed: of all the S. Typhimurium mutant strains tested, only an mgtC mutant strain partially reproduced the phenotype of the phoP mutant strain. As this gene is required for bacterial growth in magnesium-depleted conditions in vitro, the contributions of PhoP/Q to intramacrophage replication and survival were reappraised. Although PhoP/Q was required for both replication and survival of S. Typhimurium within murine macrophages, subsequent analysis of the kinetics of phagolysosomal fusion, taking account of differences in the replication rates of wild-type and phoP mutant strains, provided no evidence for a PhoP/Q-dependent role in this process. PhoP/Q appeared to act subsequent to the process of phagolysosomal avoidance and to promote replication of those bacteria that had already escaped a phagolysosomal fate. Therefore, we conclude that the PhoP/Q regulon enables S. Typhimurium to adapt to intramacrophage stresses other than phagolysosomal fusion

DC-SIGN and DC-SIGNR Bind Ebola Glycoproteins and Enhance Infection of Macrophages and Endothelial Cells

AbstractEbola virus exhibits a broad cellular tropism in vitro. In humans and animal models, virus is found in most tissues and organs during the latter stages of infection. In contrast, a more restricted cell and tissue tropism is exhibited early in infection where macrophages, liver, lymph node, and spleen are major initial targets. This indicates that cellular factors other than the broadly expressed virus receptor(s) modulate Ebola virus tropism. Here we demonstrate that the C-type lectins DC-SIGN and DC-SIGNR avidly bind Ebola glycoproteins and greatly enhance transduction of primary cells by Ebola virus pseudotypes and infection by replication-competent Ebola virus. DC-SIGN and DC-SIGNR are expressed in several early targets for Ebola virus infection, including dendritic cells, alveolar macrophages, and sinusoidal endothelial cells in the liver and lymph node. While DC-SIGN and DC-SIGNR do not directly mediate Ebola virus entry, their pattern of expression in vivo and their ability to efficiently capture virus and to enhance infection indicate that these attachment factors can play an important role in Ebola transmission, tissue tropism, and pathogenesis