Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

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A robust method for particulate detection of a genetic tag for 3D electron microscopy

eLife ◽

10.7554/elife.64630 ◽

2021 ◽

Vol 10 ◽

Author(s):

James Rae ◽

Charles Ferguson ◽

Nicholas Ariotti ◽

Richard I Webb ◽

Han-Hao Cheng ◽

...

Keyword(s):

Electron Microscopy ◽

Fusion Proteins ◽

Ion Beam ◽

Expression System ◽

Internal Standard ◽

Membrane Vesicles ◽

Cytoskeletal Proteins ◽

Electron Microscopic ◽

Simple Method ◽

In Cells

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

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Gastric Ulcers Produced by Cisplatin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099337 ◽

1981 ◽

Vol 39 ◽

pp. 582-583

Author(s):

S.K. Aggarwal ◽

J. San Antonio

Keyword(s):

Electron Microscopy ◽

Single Dose ◽

Side Effects ◽

Antitumor Agent ◽

Gastric Ulcers ◽

Enzyme Cytochemistry ◽

Intestinal Toxicity ◽

Ovarian Cancers ◽

Inner Surface

Cisplatin (cis-dichlorodiammineplatinum(II)) a potent antitumor agent is now available for the treatment of testicular and ovarian cancers. It is however, not free from its serious side effects including nephrotoxicity, gastro intestinal toxicity, myelosuppression, and ototoxicity. Here we now report that the drug produces peculiar bloating of the stomach in rats and induces acute ulceration.Wistar-derived rats weighing 200-250 g were administered cisplatin(9 mg/kg) ip as a single dose in 0.15 M NaCl. After 3 days the animals were sacrificed by decapitation. The stomachs were removed, the contents analyzed for pepsin and acidity. The inner surface was examined with a dissecting microscope after a moderate stretching for ulcers. Affected areas were fixed and processed for routine electron microscopy and enzyme cytochemistry.The drug treated animals kept on food and water consistently showed bloating and lesions (Fig. 1) with a frequency of 6-70 ulcers in the rumen section of the stomachs.

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Structure and Function of Major SARS-CoV-2 and SARS-CoV Proteins

Bioinformatics and Biology Insights ◽

10.1177/11779322211025876 ◽

2021 ◽

Vol 15 ◽

pp. 117793222110258

Author(s):

Ritesh Gorkhali ◽

Prashanna Koirala ◽

Sadikshya Rijal ◽

Ashmita Mainali ◽

Adesh Baral ◽

...

Keyword(s):

Genomic Organization ◽

Membrane Vesicles ◽

Structural Proteins ◽

Host Immune System ◽

Accessory Proteins ◽

Nonstructural Proteins ◽

N Protein ◽

Small Proteins ◽

Methylation Mark ◽

And Function

SARS-CoV-2 virus, the causative agent of COVID-19 pandemic, has a genomic organization consisting of 16 nonstructural proteins (nsps), 4 structural proteins, and 9 accessory proteins. Relative of SARS-CoV-2, SARS-CoV, has genomic organization, which is very similar. In this article, the function and structure of the proteins of SARS-CoV-2 and SARS-CoV are described in great detail. The nsps are expressed as a single or two polyproteins, which are then cleaved into individual proteins using two proteases of the virus, a chymotrypsin-like protease and a papain-like protease. The released proteins serve as centers of virus replication and transcription. Some of these nsps modulate the host’s translation and immune systems, while others help the virus evade the host immune system. Some of the nsps help form replication-transcription complex at double-membrane vesicles. Others, including one RNA-dependent RNA polymerase and one exonuclease, help in the polymerization of newly synthesized RNA of the virus and help minimize the mutation rate by proofreading. After synthesis of the viral RNA, it gets capped. The capping consists of adding GMP and a methylation mark, called cap 0 and additionally adding a methyl group to the terminal ribose called cap1. Capping is accomplished with the help of a helicase, which also helps remove a phosphate, two methyltransferases, and a scaffolding factor. Among the structural proteins, S protein forms the receptor of the virus, which latches on the angiotensin-converting enzyme 2 receptor of the host and N protein binds and protects the genomic RNA of the virus. The accessory proteins found in these viruses are small proteins with immune modulatory roles. Besides functions of these proteins, solved X-ray and cryogenic electron microscopy structures related to the function of the proteins along with comparisons to other coronavirus homologs have been described in the article. Finally, the rate of mutation of SARS-CoV-2 residues of the proteome during the 2020 pandemic has been described. Some proteins are mutated more often than other proteins, but the significance of these mutation rates is not fully understood.

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ATP-linked calcium transport in cells and membrane vesicles of Streptococcus faecalis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)38043-2 ◽

1978 ◽

Vol 253 (7) ◽

pp. 2085-2092 ◽

Cited By ~ 26

Author(s):

H. Kobayashi ◽

J. Van Brunt ◽

F.M. Harold

Keyword(s):

Calcium Transport ◽

Membrane Vesicles ◽

Streptococcus Faecalis ◽

In Cells

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Revisiting staining of biological samples for electron microscopy: perspectives for recent research

Materials Horizons ◽

10.1039/d0mh01579b ◽

2021 ◽

Author(s):

Maren T. Kuchenbrod ◽

Ulrich S. Schubert ◽

Rainer Heintzmann ◽

Stephanie Hoeppener

Keyword(s):

Electron Microscopy ◽

Biological Samples ◽

Active Sites ◽

In Cells

This review revisits staining protocols for electron microscopy focussing on the visualization of active sites, i.e. enzymes, metabolites or proteins, in cells and tissues, which were never established as standard protocols in electron microscopy.

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Emerging viruses – a challenge to diagnostic Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927603041102 ◽

2003 ◽

Vol 9 (S03) ◽

pp. 522-523

Author(s):

Dirk Soike

Keyword(s):

Electron Microscopy ◽

Emerging Viruses

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Structure and assembly of double-headed Sendai virus nucleocapsids

Communications Biology ◽

10.1038/s42003-021-02027-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Na Zhang ◽

Hong Shan ◽

Mingdong Liu ◽

Tianhao Li ◽

Rui Luo ◽

...

Keyword(s):

Electron Microscopy ◽

Measles Virus ◽

Sendai Virus ◽

Nipah Virus ◽

Mumps Virus ◽

Genome Replication ◽

Direct Visualization ◽

Closed State ◽

Cryo Electron Microscopy ◽

Negative Sense

AbstractParamyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

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