Preparation of vaccine against RNA virus e.g. picornavirus, orthomyxovirus, retrovirus comprises infecting BHK21 cell culture, separating intact cell nuclei from cytoplasmic fraction and precipitating antigenic proteins

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a human-liver-specific microRNA, termed miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) of the viral genome, and this interaction promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been demonstrated to be efficacious in reducing HCV titers in chronic HCV-infected patients. However, the precise mechanism(s) of miR-122-mediated viral RNA accumulation have remained elusive. We have used biophysical analysis and assays for viral replication in cell culture to understand the interactions between the human Argonaute 2 (hAgo2):miR-122 complex and the HCV genome. In addition, we have analyzed several resistance-associated variants which were isolated from patients who underwent miR-122 inhibitor-based therapy in order to shed light on novel mechanisms of antiviral resistance. Our results provide a new model for miR-122:HCV RNA interactions and demonstrate that miR-122 plays at least three roles in the HCV life cycle: (1) miR-122 acts as an RNA chaperone to suppress an energetically favorable secondary structure and allows the viral internal ribosomal entry site (IRES) to form; (2) miR-122 binding to the 5' terminus protects the genome from the activity of cellular pyrophosphatases (DOM3Z and DUSP11) and subsequent exonuclease-mediated decay; and (3) the Argonaute (Ago) protein at Site 2 makes direct contact with the HCV IRES, enhancing viral translation. In addition, analyses of several resistance-associated variants that were isolated from patients that underwent miR-122 inhibitor-based therapy suggests that mutations in the 5' terminus alter the structure of the 5' UTR in a manner that promotes RNA chaperone activity or viral genome stability, even in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and suggest new mechanisms of antiviral resistance which are mediated by changes in RNA structure.

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Surface modification of uniaxial cyclic strain cell culture platform with temperature-responsive polymer for cell sheet detachment

Journal of Materials Chemistry B ◽

10.1039/c5tb01171j ◽

2015 ◽

Vol 3 (40) ◽

pp. 7899-7902 ◽

Cited By ~ 2

Author(s):

E. L. Lee ◽

H. H. Bendre ◽

A. Kalmykov ◽

J. Y. Wong

Keyword(s):

Cell Culture ◽

Intact Cell ◽

Cell Sheet ◽

Cyclic Strain ◽

Cell Phenotype ◽

Temperature Responsive ◽

Current Cell ◽

Responsive Polymer ◽

Smooth Muscle Cell Phenotype ◽

A Cell

Because current cell sheet-based blood vessels lack biomimetic structure and require excessively long culture times that may compromise smooth muscle cell phenotype, we modified a cell culture platform with thermoresponsive copolymers to allow intact cell sheet detachment after uniaxial conditioning.

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Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra118.000924 ◽

2018 ◽

Vol 17 (10) ◽

pp. 2018-2033 ◽

Cited By ~ 34

Author(s):

Domenico Fasci ◽

Hugo van Ingen ◽

Richard A. Scheltema ◽

Albert J. R. Heck

Keyword(s):

Mass Spectrometry ◽

Intact Cell ◽

Cell Nuclei

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Progesterone receptor does not form oligomeric (8S), non-DNA-binding complex in intact cell nuclei

Journal of Cellular Biochemistry ◽

10.1002/jcb.240580112 ◽

1995 ◽

Vol 58 (1) ◽

pp. 95-104 ◽

Cited By ~ 8

Author(s):

Anu Pekki ◽

Timo Ylikomi ◽

Heimo Syvälä ◽

Pentti Tuohimaa

Keyword(s):

Dna Binding ◽

Progesterone Receptor ◽

Intact Cell ◽

Cell Nuclei

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Synthesis of adenosine triphosphate in isolated nuclei and intact cells

Biochemical Journal ◽

10.1042/bj1020878 ◽

1967 ◽

Vol 102 (3) ◽

pp. 878-884 ◽

Cited By ~ 12

Author(s):

H. M. Klouwen ◽

A. W. M. Appelman

Keyword(s):

Oxidative Phosphorylation ◽

Intact Cell ◽

Atp Synthesis ◽

Lymphoid Cells ◽

Cell Nuclei ◽

Isolated Cells ◽

Intact Cells ◽

Isolated Nuclei ◽

Janus Green ◽

Stimulation Of

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1-10mum) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under ;nuclear' conditions and in the presence of Janus Green B and Ca(2+) is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.

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An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor Galidesivir (BCX4430) and also attenuates the virus for mice

10.1101/563544 ◽

2019 ◽

Cited By ~ 2

Author(s):

Ludek Eyer ◽

Antoine Nougairède ◽

Marie Uhlířová ◽

Jean-Sélim Driouich ◽

Darina Zouharová ◽

...

Keyword(s):

Cell Culture ◽

Ebola Virus ◽

Rna Virus ◽

Encephalitis Virus ◽

Viral Fitness ◽

Rna Dependent Rna Polymerase ◽

Virus Inhibitor ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus ◽

Ebola Virus Infection

AbstractThe adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell culture. Stochastic molecular simulations indicate that the steric freedom caused by the E460D substitution increases close electrostatic interactions between the inhibitor and the interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an incorrect/modified nucleotide. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2’-C-methyladenosine, 2’-C-methyladenosine and 4’-azido-aracytidine. Although, the E460D substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to flavivirus neurovirulence.ImportanceTick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. We have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola virus infection, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to Galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of viral RNA genome. Although, this substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity.

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miR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103671118 ◽

2021 ◽

Vol 118 (33) ◽

pp. e2103671118

Author(s):

Jasmin Chahal ◽

Luca F. R. Gebert ◽

Carolina Camargo ◽

Ian J. MacRae ◽

Selena M. Sagan

Keyword(s):

Cell Culture ◽

Rna Structure ◽

Genome Stability ◽

Rna Virus ◽

Resistance Mechanisms ◽

Viral Rna ◽

Hcv Rna ◽

Chronic Hcv ◽

Rna Accumulation ◽

Insight Into

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5′ untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor–based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122–mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

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Electron Microscopic Analysis of active and Repressed Chromatin within Normal and Neoplastic Human Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002397 ◽

1980 ◽

Vol 38 ◽

pp. 542-543

Author(s):

J. H. Frenster

Keyword(s):

Rna Synthesis ◽

Molecular Conformation ◽

Intact Cell ◽

Microscopic Analysis ◽

Dna Molecules ◽

Cell Nuclei ◽

Animal Cells ◽

Electron Microscopic ◽

Acidic Proteins ◽

Dna Template

Chromatin within the nuclei of animal cells consists of DNA molecules in non-covalent association with such intra-nuclear macromolecules as histones, acidic proteins, lipoproteins, and RNA species (1). These associated ligands and counter-ligands direct the molecular conformation and biological activity of DNA molecules within intact cell nuclei and within the intact chromatin complexes that can be isolated from such cell nuclei (2). Two major phase states of chromatin are recognized by ultrastructural, bio-synthetic, and biophysical criteria, and these two states of chromatin contain DNA template molecules that are either active in or repressed for RNA synthesis (3). DNA template molecules active in RNA synthesis are found within extended euchromatin microfibrils (Chart 1) which contain increased amounts and kinds of acidic proteins, lipoproteins, and RNA species. Each of these molecular species Is capable of activating for RNA synthesis the DNA molecules within repressed heterochromatin (Chart 1). By contrast, the DNA molecules within condensed heterochromatin are found to be repressed for RNA synthesis (3), and such repressed heterochromatin is relatively devoid of acidic proteins, lipoproteins, and RNA species.

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Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei

Journal of Cell Science ◽

10.1242/jcs.115.10.2165 ◽

2002 ◽

Vol 115 (10) ◽

pp. 2165-2177

Author(s):

Khristy J. Thompson ◽

Michael G. Fried ◽

Zheng Ye ◽

Phillip Boyer ◽

James R. Connor

Keyword(s):

Cell Culture ◽

Oxidative Damage ◽

Biological Significance ◽

Nuclear Pore ◽

Cell Nuclei ◽

Iron Storage ◽

Cellular Iron ◽

Human Astrocytoma ◽

Culture Models

Ferritin is traditionally considered a cytoplasmic iron-storage protein,but recent reports indicate that it is also found in cell nuclei. Nuclear ferritin has been proposed to be involved in both the protection of DNA and the exacerbation of iron-induced oxidative damage to DNA. We demonstrate that H-rich ferritin is present in the nucleus of human astrocytoma tumor cells. To study the mechanism and regulation of ferritin translocation to the nucleus,we developed a cell culture model using SW1088 human astrocytoma cells. Changes in cellular iron levels, cytokine treatments and hydrogen peroxide exposure affected the distribution of ferritin between the cytosol and the nucleus. Ferritin enters the nucleus via active transport through the nuclear pore and does not require NLS-bearing cytosolic factors for transport. Furthermore, H-rich ferritin is preferred over L-rich ferritin for uptake into the nucleus. Whole cell crosslinking studies revealed that ferritin is associated with DNA. Ferritin protected DNA from iron-induced oxidative damage in both in vitro and in cell culture models. These results strongly suggest a novel role for ferritin in nuclear protection. This work should lead to novel characterization of ferritin functions in the context of genomic stability and may have unparalleled biological significance in terms of the accessibility of metals to DNA. The knowledge generated as a result of these studies will also improve our understanding of iron-induced damage of nuclear constituents.

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Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy

Biochemical Society Transactions ◽

10.1042/bst0310071 ◽

2003 ◽

Vol 31 (1) ◽

pp. 71-75 ◽

Cited By ~ 7

Author(s):

H. Oberleithner ◽

C. Schäfer ◽

V. Shahin ◽

L. Albermann

Keyword(s):

Atomic Force Microscopy ◽

Molecular Mass ◽

Intact Cell ◽

Rnase A ◽

Nuclear Pore ◽

Xenopus Laevis Oocyte ◽

Cell Nuclei ◽

Homogeneous Population ◽

Force Microscopy ◽

Atomic Force

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

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