Oncogenic virus (tumor virus)

AbstractPolyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposi’s sarcoma associated Herpesvirus (KSHV) is a defined oncogenic virus belonging to the sub-family of human gamma-herpesviruses. KSHV infection leads to the profound alteration of host metabolic landscape to favor the development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection likely through the modulation of key enzymes of these pathways, such as ODC1, and that in return these metabolic pathways are required for both KSHV lytic switch from latency and de novo infection. The further analysis unraveled that translation of critical KSHV latent and lytic proteins (LANA, RTA) depends on eIF5A hypusination. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by using inhibitors targeting either polyamine biosynthesis or eIF5A hypusination. Above all, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination metabolic pathways promote viral propagation and oncogenesis, which serve as new therapeutic targets to treat KSHV-associated malignancies.

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Electron Microscopy of the Nucleic Acid of Mouse Mammary Tumor Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099854 ◽

1968 ◽

Vol 26 ◽

pp. 22-23

Author(s):

N. H. Sarkar ◽

Dan H. Moore

Keyword(s):

Mammary Tumor ◽

Mouse Mammary Tumor Virus ◽

Ammonium Acetate ◽

Dry Weight ◽

Mouse Tumor ◽

Mammary Tumor Virus ◽

Rna Molecules ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

The Subject

Mouse mammary tumor virus (MTV) is believed to contain about 0.8% single stranded ribonucleic acid (RNA). This value of RNA content was estimated on a dry weight basis. The subject of this report is an attempt to visualize the RNA molecules of MTV particles.MTV particles were isolated from RIII mouse (tumor incidence approximately 80%) milk according to the method described by Lyons and Moore. Purified virions from 5 ml of milk were finally suspended in 0.2 ml of PBS, pH 7.4 and was mixed with an equal volume of pronase (5 mg/ml). This mixture was incubated at 37°C for an hour. RNA was extracted three times using freshly prepared cold phenol. It was then treated three times with cold ethyl ether to remove any trace of phenol. The RNA thus extracted was divided into two parts. One part was diluted four fold with 8M urea to avoid aggregation of the molecules. The other part was left untreated. Both samples were then mixed with an equal volume of 1M ammonium acetate, adjusted to pH 8.0 with NH3 containing chymotrypsin at a concentration of 0.01%.

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Electron Immunochemical Labeling of Ultrathin Sections of Murine Tumor Viruses and Cell Cultures using the Unlabeled Antibody Enzyme Technique

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100091317 ◽

1976 ◽

Vol 34 ◽

pp. 266-267

Author(s):

W. J. Hamilton

Keyword(s):

Mammary Tumor ◽

Leukemia Virus ◽

Parallel Methods ◽

Tumor Viruses ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Antibody Conjugates ◽

Rauscher Leukemia ◽

Antigen Localization ◽

Unlabeled Antibody

The study of RNA tumor viruses has been greatly facilitated by the use of immunochemical tagging methods. In the past these methods have been constrained to antibody conjugates with ferritin or peroxidase. In order to avoid the disadvantages of using conjugated antisera, investigators have applied the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells prior to embedding for electron microscopy. The current study has successfully applied the Sternberger method to virusproducing cells and purified virus pellets after epoxy-embedding and ultrathin sectioning. The results demonstrate the distinct advantages of this “post-embedding” method for viral antigen localization.Purified Rauscher leukemia virus (RLV) and mouse mammary tumor virus (MMTV) were pelleted, fixed in buffered 2% paraformaldehyde and washed thoroughly. These were dehydrated in acetone, infiltrated and embedded in Spurr resin according to common procedures. A tumor derived cell line, Mm5mt, producing MMTV was embedded by parallel methods.

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Lectin Binding Studies on RNA Tumor Virus-Infected Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115250 ◽

1975 ◽

Vol 33 ◽

pp. 326-327

Author(s):

D. C. Hixson

Keyword(s):

Binding Sites ◽

Lectin Binding ◽

Culture Conditions ◽

3T3 Cells ◽

Binding Studies ◽

Con A ◽

Microscopic Studies ◽

Tumor Virus ◽

Infected Cells ◽

Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

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Detection of Viral Antigens in Mouse and Rat Tumor Cells by Immunoelectron Microscopy Following Periodate-Lysine-Paraformaldehyde (PLP) Fixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009124x ◽

1976 ◽

Vol 34 ◽

pp. 252-253

Author(s):

Y. Ohtsuki ◽

G. Seman ◽

J. M. Bowen ◽

M. Scanlon ◽

L. Dmochowski

Keyword(s):

Immunoelectron Microscopy ◽

Mammary Tumor ◽

Type A ◽

Rabbit Serum ◽

Virus Particles ◽

Viral Antigens ◽

Culture Cells ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Immunoperoxidase Labeling

Recently, periodate-lysine-paraformaldehyde (PLP) fixation was reported for immunoelectron microscopy (1). In PLP fixation, carbohydrates are oxidized by periodate and cross-linked by lysine; paraformaldehyde stabilizes proteins and lipids. By using PLP fixation, intracytoplasmic type A viral antigens have been previously demonstrated by immunoperoxidase labeling (2). In the present study, PLP fixation has been applied for the detection of the same antigens in mouse mammary tumor culture cells by both immunoferritin and immunoperoxidase methods. Rabbit anti-intracytoplasmic type A virus serum (anti-A), kindly provided by Dr. M. Muller (3), rabbit anti-strain A mouse mammary tumor virus (anti-MMTV) and preimmune rabbit serum as control were used to detect viral antigens in cells of C3H/HeJ strain mouse mammary tumor culture. Attempts have been also made to demonstrate peroxidase labeling of type C virus particles in frozen sections of an SD-MSV-induced NZB rat bone tumor tissue by rabbit anti-MuLV serum.

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Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115572 ◽

1975 ◽

Vol 33 ◽

pp. 390-391

Author(s):

Wm. J. Arnold ◽

J. Russo ◽

H. D. Soule ◽

M. A. Rich

Keyword(s):

Horseradish Peroxidase ◽

Mammary Tumor ◽

Covalent Binding ◽

Petri Dish ◽

Gamma Chain ◽

Mammary Tumor Virus ◽

Mammary Tumor Cells ◽

Murine Mammary Tumor ◽

Tumor Virus ◽

Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

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Immunoferritin Labelling of Murine Leukemia Virus Antigens in thin Sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002221 ◽

1980 ◽

Vol 38 ◽

pp. 508-509

Author(s):

Ray A. Weigand ◽

Gregory C. Varjabedian

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Intracellular Localization ◽

Uranyl Acetate ◽

Antibody Technique ◽

Thin Sections ◽

Tumor Virus ◽

Labelling Procedure ◽

Unlabelled Antibody ◽

Murine Leukemia

We previously described the intracellular localization of murine mammary tumor virus (MuMTV) p28 protein in thin sections (1). In that study, MuMTV containing cells fixed in 3% paraformaldehyde plus 0.05% glutaraldehyde were labelled after thin sectioning using ferritin-antiferritin in an unlabelled antibody technique. We now describe the labelling of murine leukemia virus (MuLV) particles using the unlabelled antibody technique coupled to ferritin-Fab antiferritin. Cultures of R-MuLV in NIH/3T3 cells were grown to 90% confluence (2), fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M cacodylate at pH 7.2, postfixed with buffered 17 OsO4, dehydrated with a series of etha-nols, and embedded in Epon. Thin sections were collected on nickel grids, incubated in 107 H2O2, rinsed in HEPES buffered saline, and subjected to the immunoferritin labelling procedure. The procedure included preincubation in 27 egg albumin, a four hour incubation in goat antisera against purified gp69/71 of MuLV (3) (primary antibody), incubation in F(ab’)2 fragments of rabbit antisera to goat IgG (secondary antibody), incubation in apoferritin, incubation in ferritin-Fab ferritin, and a brief fixation with 2% glutaraldehyde. The sections were stained with uranyl acetate and examined in a Siemens IA electron microscope at an accelerating voltage of 60 KV.

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