A 3.4-Å cryo-electron microscopy structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles

Abstract Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV-1 and SARS-CoV-2. Here, we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a three-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other sites, as well as different glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies or drugs, in the native state of the coronavirus particles.

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A 3.4-Å cryo-EM structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles

10.1101/2020.08.11.245696 ◽

2020 ◽

Author(s):

Kaiming Zhang ◽

Shanshan Li ◽

Grigore Pintilie ◽

David Chmielewski ◽

Michael F. Schmid ◽

...

Keyword(s):

Viral Entry ◽

Respiratory Diseases ◽

Respiratory Illness ◽

Human Coronavirus ◽

Future Studies ◽

Virus Particles ◽

Glycosylation Sites ◽

The Family ◽

Upper Respiratory

AbstractHuman coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other amino acid positions as well as differences in glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the coronavirus particles.

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SARS-CoV-2 Infection in the Central Nervous System of a 1-Year-Old Infant Submitted to Complete Autopsy

10.20944/preprints202009.0297.v1 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ismael Carlos Gomes ◽

Karina Karmirian ◽

Julia Oliveira ◽

Carolina Pedrosa ◽

Fernando Colonna Rosman ◽

...

Keyword(s):

Respiratory Illness ◽

Organ Damage ◽

Multiple Organ ◽

Post Mortem ◽

Cerebral Damage ◽

Virus Particles ◽

Brain Infection ◽

Post Mortem Brain ◽

The Central Nervous System

Coronavirus disease 2019 (COVID-19) was initially characterized as a respiratory illness. Neurological manifestations were reported mostly in severely affected patients. Routes for brain infection and the presence of virus particles in situ have not been well described, raising controversy about how the virus causes neurological symptoms. Here, we report the autopsy findings of a 1-year old infant with COVID-19. In addition to pneumonitis, meningitis and multiple organ damage related to thrombosis, a previous encephalopathy may have contributed to additional cerebral damage. SARS-CoV-2 infected the choroid plexus, ventricles, and cerebral cortex. This is the first evidence of SARS-CoV-2 detection in an infant post-mortem brain.

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Structure, receptor recognition and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein

10.1101/2021.10.25.465646 ◽

2021 ◽

Author(s):

M. Alejandra Tortorici ◽

Alexandra C Walls ◽

Anshu Joshi ◽

Young-Jun Park ◽

Rachel T Eguia ◽

...

Keyword(s):

Viral Entry ◽

Nucleotide Polymorphisms ◽

Spike Glycoprotein ◽

Human Coronavirus ◽

Single Nucleotide ◽

Zoonotic Pathogen ◽

Cryo Electron Microscopy ◽

Receptor Recognition ◽

Therapeutic Development ◽

Entry Receptor

The recent isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. Here, we determined cryo-electron microscopy structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and identified that it binds canine, feline and porcine aminopeptidase N (APN encoded by ANPEP) orthologs which serve as entry receptors. Introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single nucleotide polymorphisms could account for the detection of this virus in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 S-mediated entry, indicating elicitation of cross-neutralizing activity among α-coronaviruses. These data provide a blueprint of the CCoV-HuPn-2018 infection machinery, unveil the viral entry receptor and pave the way for vaccine and therapeutic development targeting this zoonotic pathogen.

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Electron Microscopy of Mycoplasma Pneumoniae

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065341 ◽

1971 ◽

Vol 29 ◽

pp. 258-259 ◽

Cited By ~ 1

Author(s):

E. S. Boatman ◽

G. E. Kenny

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Growth Phase ◽

Hela Cells ◽

Sulfonic Acid ◽

Gamma Globulin ◽

Horse Serum ◽

Morphological Aspects ◽

The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

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Exposure of protein VP7 on the surface of bluetongue virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160558 ◽

1990 ◽

Vol 48 (3) ◽

pp. 600-601

Author(s):

A.D. Hyatt

Keyword(s):

Bluetongue Virus ◽

Structural Proteins ◽

Major Constituent ◽

Outer Coat ◽

Virus Particles ◽

Antibody Recognition ◽

The Core ◽

The Family ◽

Electron Microscopical ◽

Physical Accessibility

Bluetongue virus (BTV) is the type species os the genus orbivirus in the family Reoviridae. The virus has a fibrillar outer coat containing two major structural proteins VP2 and VP5 which surround an icosahedral core. The core contains two major proteins VP3 and VP7 and three minor proteins VP1, VP4 and VP6. Recent evidence has indicated that the core comprises a neucleoprotein center which is surrounded by two protein layers; VP7, a major constituent of capsomeres comprises the outer and VP3 the inner layer of the core . Antibodies to VP7 are currently used in enzyme-linked immunosorbant assays and immuno-electron microscopical (JEM) tests for the detection of BTV. The tests involve the antibody recognition of VP7 on virus particles. In an attempt to understand how complete viruses can interact with antibodies to VP7 various antibody types and methodologies were utilized to determine the physical accessibility of the core to the external environment.

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Faculty Opinions recommendation of Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1144933.602036 ◽

2009 ◽

Author(s):

William Earnshaw

Keyword(s):

Electron Microscopy ◽

Mitotic Chromosomes ◽

Cryo Electron Microscopy ◽

Chromatin Fibers ◽

Microscopy Images

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Species-diagnostic and species-specific DNA sequences evenly distributed throughout pine and spruce chromosomes

Genome ◽

10.1139/g10-065 ◽

2010 ◽

Vol 53 (10) ◽

pp. 769-777 ◽

Cited By ~ 1

Author(s):

Melanie Mehes-Smith ◽

Paul Michael ◽

Kabwe Nkongolo

Keyword(s):

Dna Sequences ◽

Random Amplified Polymorphic Dna ◽

Inter Simple Sequence Repeat ◽

Issr Markers ◽

Pinus Monticola ◽

The Family ◽

Species Specific ◽

Rapd And Issr ◽

Specific Sequences

Genome organization in the family Pinaceae is complex and largely unknown. The main purpose of the present study was to develop and physically map species-diagnostic and species-specific molecular markers in pine and spruce. Five RAPD (random amplified polymorphic DNA) and one ISSR (inter-simple sequence repeat) species-diagnostic or species-specific markers for Picea mariana , Picea rubens , Pinus strobus , or Pinus monticola were identified, cloned, and sequenced. In situ hybridization of these sequences to spruce and pine chromosomes showed the sequences to be present in high copy number and evenly distributed throughout the genome. The analysis of centromeric and telomeric regions revealed the absence of significant clustering of species-diagnostic and species-specific sequences in all the chromosomes of the four species studied. Both RAPD and ISSR markers showed similar patterns.

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Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function

Genes ◽

10.3390/genes11121511 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1511

Author(s):

Tatyana V. Karamysheva ◽

Tatyana A. Gayner ◽

Vladimir V. Muzyka ◽

Konstantin E. Orishchenko ◽

Nikolay B. Rubtsov

Keyword(s):

Genetic Counseling ◽

Chromosome Rearrangement ◽

Marker Chromosome ◽

Reproductive Function ◽

Small Supernumerary Marker Chromosome ◽

Comprehensive Chromosome Screening ◽

The Family ◽

Multicolor Banding ◽

The One

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.

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