scholarly journals Neuropilin-1 is a host factor for SARS-CoV-2 infection

Science ◽  
2020 ◽  
Vol 370 (6518) ◽  
pp. 861-865 ◽  
Author(s):  
James L. Daly ◽  
Boris Simonetti ◽  
Katja Klein ◽  
Kai-En Chen ◽  
Maia Kavanagh Williamson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Interference ◽  
Cell Attachment ◽  
Host Factor ◽  
Terminal Sequence ◽  
X Ray ◽  
X Ray Crystallography ◽  
Neuropilin 1 ◽  
Host Cell Attachment ◽  
Carboxyl Terminal

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

scholarly journals Neuropilin-1 is a host factor for SARS-CoV-2 infection

2020 ◽  
Author(s):  
James L. Daly ◽  
Boris Simonetti ◽  
Carlos Antón-Plágaro ◽  
Maia Kavanagh Williamson ◽  
Deborah K. Shoemark ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Attachment ◽  
Host Factor ◽  
Sequence Motif ◽  
Terminal Sequence ◽  
Viral Glycoprotein ◽  
Specific Sequence ◽  
S Protein ◽  
Neuropilin 1 ◽  
Host Cell Attachment

SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(3,4). For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors5. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.

scholarly journals [Ag(I)(Et2PCH2CH2PPh2)2]NO3: An Antimitochondrial Silver Complex

1995 ◽  
Vol 2 (2) ◽  
pp. 111-122 ◽  
Author(s):  
Susan J. Berners-Price ◽  
Doreen C. Collier ◽  
Muhammed A. Mazid ◽  
Peter J. Sadler ◽  
Rodney E. Sue ◽  
...  
Keyword(s):  
Cell Culture ◽  
Serum Albumin ◽  
Culture Media ◽  
Oxidized Glutathione ◽  
Cell Culture Media ◽  
Mitochondrial Mutation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Yeast Strains ◽  
Screening Protocol

The silver(I) complex [Ag(eppe)2]NO3 (eppe = Et2PCH2CH2PPh2) is shown by X-ray crystallography to be tetrahedral with Ag - PEt2 and Ag - P Ph2 bond lengths of 2.482 and 2.518 Å, respectively. The complex is selectively antimitochondrial and inhibits the growth of a number of yeast strains in non-fermentable media at concentrations as low as 2.5 μΜ and induces the mitochondrial mutation petite The effect is largely reversed by the presence of aspirin. The complex is shown to be stable in the cell culture media and in the presence of glutathione, but readily reacts with disulfides of oxidized glutathione and serum albumin. Surprisingly, neither [Au(eppe)2]Cl nor [Au(dppe)2]Cl (dppe = Ph2PCH2CH2PPh2) showed any mitochondrial selectivity in the same screening protocol.

scholarly journals Structure of SARS-CoV-2 ORF8, a rapidly evolving coronavirus protein implicated in immune evasion

2020 ◽  
Author(s):  
Thomas G. Flower ◽  
Cosmo Z. Buffalo ◽  
Richard M. Hooy ◽  
Marc Allaire ◽  
Xuefeng Ren ◽  
...  
Keyword(s):  
Immune Responses ◽  
Immune Evasion ◽  
Immune Suppression ◽  
Molecular Basis ◽  
Large Scale ◽  
Terminal Sequence ◽  
Specific Sequence ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rapid Spread

AbstractThe molecular basis for the severity and rapid spread of the COVID-19 disease caused by SARS-CoV-2 is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04 Å resolution by x-ray crystallography. The structure reveals a ~60 residue core similar to SARS-CoV ORF7a with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate non-covalent interface is formed by another SARS-CoV-2-specific sequence, 73YIDI76. Together the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.

Dihydrofolate Reductase Inhibition. A Study in the Use of X-ray Crystallography, Molecular Graphics, and Quantitative Structure-Activity Relations in Drug Design

1982 ◽  
Vol 16 (5) ◽  
pp. 391-396 ◽  
Author(s):  
Corwin Hansch
Keyword(s):  
Cell Culture ◽  
Dihydrofolate Reductase ◽  
Three Dimensional ◽  
Bovine Liver ◽  
Quantitative Structure ◽  
Molecular Graphics ◽  
X Ray ◽  
X Ray Crystallography ◽  
Structure Activity ◽  
Hydrophobic Barrier

Substituent constants and regression analyses are used to formulate quantitative structure-activity relationships (QSAR) for the inhibition by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-X-phenyl)- s-triazines of purified dihydrofolate reductase (DHFR) from L. casei cells, bovine liver, and murine leukemia cells (L5178Y). The QSAR for the activity of the triazines on purified DHFR is compared with the QSAR for their action on L. casei cell culture and murine L5178Y cell culture. The QSAR for action on purified DHFR is similar to that on wild type cells; however, the QSAR for these cells differs remarkably from QSAR for both types of cells that are resistant to methotrexate (MTX). The conclusion from these analyses is that cells resistant to MTX protect themselves from this highly hydrophilic drug by developing a hydrophobic barrier. Our understanding of DHFR interaction with drugs is rapidly increasing via QSAR, and X-ray crystallography, combined with the new molecular graphics of Langridge's group, promises to expedite the process. The value of three-dimensional color graphics is discussed, with the aid of color stereo views of L. casei and E. coli DHFR.

scholarly journals CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS

2021 ◽  
Vol 118 (13) ◽  
pp. e2012898118
Author(s):  
Litao Sun ◽  
Na Wei ◽  
Bernhard Kuhle ◽  
David Blocquel ◽  
Scott Novick ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Hydrodynamic Diameter ◽  
Mutant Proteins ◽  
X Ray ◽  
Trna Synthetases ◽  
X Ray Crystallography ◽  
Charcot Marie Tooth Disease ◽  
Neuropilin 1 ◽  
Editing Domain

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.

scholarly journals Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein

2020 ◽  
Vol 118 (2) ◽  
pp. e2021785118
Author(s):  
Thomas G. Flower ◽  
Cosmo Z. Buffalo ◽  
Richard M. Hooy ◽  
Marc Allaire ◽  
Xuefeng Ren ◽  
...  
Keyword(s):  
Immune Responses ◽  
Immune Evasion ◽  
Immune Suppression ◽  
Molecular Basis ◽  
Large Scale ◽  
Terminal Sequence ◽  
Specific Sequence ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rapid Spread

The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a ∼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2−specific sequence, 73YIDI76. Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.

scholarly journals Expression des protéines VP2 et VP5 de la capside externe du virus de la fièvre catarrhale ovine

2009 ◽  
Vol 62 (2-4) ◽  
pp. 173
Author(s):  
F. Mohd Jaafar ◽  
H. Attoui ◽  
X. W. Li ◽  
O. Alpar ◽  
P. P.C. Mertens
Keyword(s):  
Cell Attachment ◽  
Animal Experiments ◽  
System Optimization ◽  
Bioinformatic Analysis ◽  
Virus Infectivity ◽  
Evolutionary Analysis ◽  
Live Attenuated Vaccine ◽  
X Ray ◽  
X Ray Crystallography ◽  
Mammalian Expression

Since 1998, nine bluetongue virus (BTV) strains from serotypes 1, 2, 4, 6, 8, 9 11, 16 and 25 have invaded Europe, killing more than two million animals (mainly sheep). Live attenuated vac­cines of BTV-2, 4, 9 and 16 have also been used in the region, in some cases causing further outbreaks of disease. Recent sequence analysis have shown that there have been cases of reassortment between field strains and live attenuated vaccine strains, hence the need for safer vaccines such as killed vaccines or recombi­nant protein-based subunit vaccines. The outer capsid protein VP2 of Culicoides-borne orbiviruses is the cell-attachment pro­tein, which carries sero-neutralization epitopes. We have cloned the open reading frame (ORF) (of segment 2) encoding VP2 and expressed the protein from BTV-4 in a bacterial expression sys­tem. The entire protein was found to be insoluble. Bioinformatic and evolutionary analysis showed that VP2 was composed of two ‘domains’, which were expressed separately using the same system. Optimization of expression conditions generated soluble proteins, indicating a correct fold of the expression products. These protein domains were used as antigens in mice experi­ments to raise antibodies against conformational epitopes. VP2 of BTV-4 was also expressed in a baculovirus system and was found to be soluble. The ORF encoding VP2 was cloned into a mammalian expression vector (pcDNA3.1) and is currently used (as a DNA vaccine) in animal experiments (using Chitosan as an adjuvant) to assess the antibody raising capacity of this formula­tion. One of the intended uses of this protein or its domains is to generate crystals for determination of the VP2 atomic structure using X-ray crystallography. VP5 of orbiviruses is a fusion pro­tein that is involved in membrane penetration during initiation of infection. The ORF (of genome segment 6) encoding VP5 of BTV-4 was cloned and expressed in the same bacterial system. Bioinformatic analysis also defined two domains of VP5. Only about 10% of the full length protein was soluble, while the two separated domains were over 90% soluble. Currently, VP5 and the two separate domains are being used in mice experiments to determine whether these can influence the immune response when concomitantly injected with the VP2 domains. VP5 was also cloned in pcDNA and used in mice experiments, formu­lated using Chitosan as an adjuvant. Antibodies collected from the mice reacted with the recombinant expressed VP5 showing high titres of antibodies. VP5 and its domains will also be used in X-ray crystallography. A plaque reduction assay was developed to determine whether the antibodies generated against VP2 domains or the VP2/VP5 mixture can neutralize virus infectivity in cell culture assays, opening the way for challenge assays in animals.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

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