scholarly journals Oncolytic H-1 Parvovirus Enters Cancer Cells through Clathrin-Mediated Endocytosis

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1199
Author(s):  
Tiago Ferreira ◽  
Amit Kulkarni ◽  
Clemens Bretscher ◽  
Karsten Richter ◽  
Marcelo Ehrlich ◽  
...  
Keyword(s):  
Viral Entry ◽  
Small Interfering Rna ◽  
Anticancer Agent ◽  
Cell Entry ◽  
Productive Infection ◽  
Acidic Ph ◽  
Coated Pits ◽  
Late Endosomes ◽  
Interfering Rna ◽  
Specific Inhibitors

H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV.

scholarly journals Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 401-407
Author(s):  
Daniel Vocelle ◽  
Olivia M Chesniak ◽  
Milton R Smith ◽  
Christina Chan ◽  
S Patrick Walton
Keyword(s):  
Small Interfering Rna ◽  
Temporal Association ◽  
Delivery Vehicle ◽  
Intracellular Location ◽  
Recycling Endosomes ◽  
Intracellular Processing ◽  
Late Endosomes ◽  
Automated Microscopy ◽  
Interfering Rna ◽  
Early Late

Abstract Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.

scholarly journals Identification of Host Factors Involved in Borna Disease Virus Cell Entry through a Small Interfering RNA Functional Genetic Screen

2010 ◽  
Vol 84 (7) ◽  
pp. 3562-3575 ◽  
Author(s):  
Roberto Clemente ◽  
Eugene Sisman ◽  
Pedro Aza-Blanc ◽  
Juan C. de la Torre
Keyword(s):  
Disease Virus ◽  
Small Interfering Rna ◽  
Cell Entry ◽  
Surface Glycoprotein ◽  
Cell Tropism ◽  
Borna Disease Virus ◽  
Virus Surface ◽  
Cellular Genes ◽  
Interfering Rna ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV), the prototypic member of the Bornaviridae family, within the order Mononegavirales, is highly neurotropic and constitutes an important model system for the study of viral persistence in the central nervous system (CNS) and associated disorders. The virus surface glycoprotein (G) has been shown to direct BDV cell entry via receptor-mediated endocytosis, but the mechanisms governing cell tropism and propagation of BDV within the CNS are unknown. We developed a small interfering RNA (siRNA)-based screening to identify cellular genes and pathways that specifically contribute to BDV G-mediated cell entry. Our screen relied on silencing-mediated increased survival of cells infected with rVSVΔG*/BDVG, a cytolytic recombinant vesicular stomatitis virus expressing BDV G that mimics the cell tropism and entry pathway of bona fide BDV. We identified 24 cellular genes involved in BDV G-mediated cell entry. Identified genes are known to participate in a broad range of distinct cellular functions, revealing a complex process associated with BDV cell entry. The siRNA-based screening strategy we have developed should be applicable to identify cellular genes contributing to cell entry mediated by surface G proteins of other viruses.

scholarly journals Zearalenone Induces Endothelial Cell Apoptosis through Activation of a Cytosolic Ca2+/ERK1/2/p53/Caspase 3 Signaling Pathway

Toxins ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 187
Author(s):  
Hyeon-Ju Lee ◽  
Se-Young Oh ◽  
Inho Jo
Keyword(s):  
Signaling Pathway ◽  
Small Interfering Rna ◽  
Caspase 3 ◽  
Cytosolic Calcium ◽  
Receptor Activation ◽  
Oxygen Species ◽  
Endothelial Cell Apoptosis ◽  
Induced Apoptosis ◽  
Interfering Rna ◽  
Specific Inhibitors

Zearalenone (ZEN) is a mycotoxin that has been reported to damage various types of cells/tissues, yet its effects on endothelial cells (ECs) have never been investigated. Therefore, this study investigates the potential effects of ZEN using bovine aortic ECs (BAECs). In this study, we found that ZEN induced apoptosis of BAECs through increased cleavage of caspase 3 and poly ADP-ribose polymerase (PARP). ZEN also increased phosphorylation of ERK1/2 and p53, and treatment with the ERK1/2 or p53 inhibitor reversed ZEN-induced EC apoptosis. Transfection of BAECs with small interfering RNA against ERK1/2 or p53 revealed ERK1/2 as an upstream target of p53 in ZEN-stimulated apoptosis. ZEN increased the production of reactive oxygen species (ROS), yet treatment with the antioxidant did not prevent EC apoptosis. Similarly, blocking of estrogen receptors by specific inhibitors also did not prevent ZEN-induced apoptosis. Finally, chelation of cytosolic calcium (Ca2+) using BAPTA-AM or inhibition of endoplasmic reticulum (ER) Ca2+ channel using 2-APB reversed ZEN-induced EC apoptosis, but not by inhibiting ER stress using 4-PBA. Together, our findings demonstrate that ZEN induces EC apoptosis through an ERK1/2/p53/caspase 3 signaling pathway activated by Ca2+ release from the ER, and this pathway is independent of ROS production and estrogen receptor activation.

scholarly journals Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol

2010 ◽  
Vol 84 (12) ◽  
pp. 6163-6176 ◽  
Author(s):  
Jeffrey W. Perry ◽  
Christiane E. Wobus
Keyword(s):  
Life Cycle ◽  
Viral Entry ◽  
Small Interfering Rna ◽  
Specific Inhibitor ◽  
Neutral Red ◽  
Dominant Negative ◽  
Murine Norovirus ◽  
Murine Macrophages ◽  
Major Route ◽  
Interfering Rna

ABSTRACT Although noroviruses cause the vast majority of nonbacterial gastroenteritis in humans, little is known about their life cycle, including viral entry. Murine norovirus (MNV) is the only norovirus to date that efficiently infects cells in culture. To elucidate the productive route of infection for MNV-1 into murine macrophages, we used a neutral red (NR) infectious center assay and pharmacological inhibitors in combination with dominant-negative (DN) and small interfering RNA (siRNA) constructs to show that clathrin- and caveolin-mediated endocytosis did not play a role in entry. In addition, we showed that phagocytosis or macropinocytosis, flotillin-1, and GRAF1 are not required for the major route of MNV-1 uptake. However, MNV-1 genome release occurred within 1 h, and endocytosis was significantly inhibited by the cholesterol-sequestering drugs nystatin and methyl-β-cyclodextrin, the dynamin-specific inhibitor dynasore, and the dominant-negative dynamin II mutant K44A. Therefore, we conclude that the productive route of MNV-1 entry into murine macrophages is rapid and requires host cholesterol and dynamin II.

scholarly journals Acquisition of Hrs, an Essential Component of Phagosomal Maturation, Is Impaired by Mycobacteria

2004 ◽  
Vol 24 (10) ◽  
pp. 4593-4604 ◽  
Author(s):  
Otilia V. Vieira ◽  
Rene E. Harrison ◽  
Cameron C. Scott ◽  
Harald Stenmark ◽  
David Alexander ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Small Interfering Rna ◽  
Early Stage ◽  
Adaptor Molecule ◽  
Phagosomal Maturation ◽  
Attachment Sites ◽  
Late Endosomes ◽  
Molecular Determinants ◽  
Inert Particles ◽  
Interfering Rna

ABSTRACT Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.

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