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Biomedical subjects

R A Consigli

Publications and source records attributed to R A Consigli.

At least 73 records · Page 4Linked to original sources
PubMed1981

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.

Acetylation↗
PubMed1980

Characterization of an extremely basic protein derived from granulosis virus nucleocapsids.

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.

Journal Article↗
PubMed1980

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella.

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.

Journal Article↗
PubMed1980

Isolation and characterization of polyoma uncoating intermediates from the nuclei of infected mouse cells.

A method was developed which enabled the efficient recovery of polyoma uncoating intermediates from the nuclei of infected cells at early times after infection (15 min to 12 h). Cells were infected with radiolabeled virus and lysed with the detergent Nonidet P-40. The nuclei were then collected and sonicated, and the products were analyzed on sucrose gradients. The uncoating intermediate sedimented at 190S and was a viral DNA-protein complex closely associated with a structure of host origin. The host material associated with the 190S uncoating intermediate was determined by polyacrylamide gel electrophoresis and visualized by electron microscopy. The amount of 190S uncoating intermediate found in the nucleus increased with time after infection. The viral DNA was predominantly for I. All of the viral proteins were present in the 190S uncoating intermediate in amounts similar to those found in viral DNA-protein complex cores.

Animals↗
PubMed1980

Separation of neutralizing and hemagglutination-inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.

Antibodies, Viral↗
PubMed1980

Effect of butylated hydroxytoluene on Newcastle disease virus.

A study was done to examine the effects of butylated hydroxytoluene (BHT) on purified Newcastle disease virus (NDV). Treatment of the virus with 50 microgram of BHT/ml caused 92% inactivation of the virion infectivity. Virion adsorption to chicken-embryonated cells was inhibited 32% and synthesis of intracellular hemagglutinin was inhibited 29%. Electron microscopy of the BHT-treated virions revealed virion envelope damage. Chicken-embryonated cells treated with 25 microgram of BHT/ml before NDV infection demonstrated 65% inhibition of NDV progeny production.

Butylated Hydroxytoluene↗
PubMed1979

In vitro reassembly of infectious polyoma virions.

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent beta-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm3 in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.

Centrifugation, Density Gradient↗
PubMed1979

Differential adsorption of polyoma virions and capsids to mouse kidney cells and guinea pig erythrocytes.

Adsorption of 125I-labeled polyoma virions and capsids to the surface of mouse kidney cells (MKC) and guinea pig erythrocytes was examined. Purified polyoma capsids lack the ability to compete with polyoma virions for specific binding sites on the surface of MKC. These same capsids were, however, able to block virion adsorption to guinea pig erythrocytes. UV-inactivated virions blocked cellular receptors on MKC and thus inhibited infectious virions from infecting the cells. Capsids were unable to inhibit virion infection of MKC. Adsorption of polyoma virions to MKC and infection of these cells were found to be independent of the ability of the virions to agglutinate guinea pig erythrocytes.

Adsorption↗
PubMed1979

Production of labile Newcastle disease virus progeny after infection of chicken embryo cells in the presence of caffeine.

A study was undertaken to examine the effects of caffeine on Newcastle disease virus (NDV) infection of chicken embryo cells. Addition of 10 mM caffeine to the growth medium produced 95% reduction in progeny synthesis, 63% reduction in RNA synthesis, 45% reduction in protein synthesis, and 25% reduction in hemadsorption ability in NDV-infected cultures when compared with untreated, infected cultures. Purified NDV obtained from caffeine-treated, infected cultures was more sensitive to ultraviolet irradiation and to damage by freezing and thawing than was observed in untreated virus cultures. The SDS-polyacrylamide gel electrophoresis revealed that purified virions contained the same complement of polypeptides, but there was a significant variation in the quantities of several of the NDV polypeptides.

Animals↗
PubMed1978

Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol.

Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.

Capsid↗
PubMed1978

Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein.

Treatment of purified polyoma virions with 6 M guanidine-hydrochloride and 0.01 M beta-mercaptoethanol resulted in the immediate loss of both hemagglutinating and plaque-forming ability. Gel filtration through Sepharose CL-6B beads allowed separation of the dimer, VP1, VP2, VP3, and histone proteins VP4-7 in highly purified form. Renaturation of the purified VP1 protein resulted in the formation of subunits that were morphologically, biophysically, and immunologically similar to native virion capsomeres.

Capsid↗
PubMed1978

Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

Animals↗
PubMed1977

Supercoiled circular DNA of an insect granulosis virus.

The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of (3)H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase. The molecular weight of the viral DNA was calculated to be 81 x 10(6) by sedimentation in neutral sucrose and 78 x 10(6) by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 x 10(6). The buoyant density of the granulosis virus DNA was 1.703 g/cm(3) and that of its insect host DNA was 1.697 g/cm(3). Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively.

Animals↗
PubMed1977

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Animals↗
PubMed1977

Immunological reactivity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

A study was undertaken to produce antisera to sodium dodecyl sulfate-derived polyoma virion polypeptides. With the use of this antisera, it was possible to detect, by immunofluorescence, cytoplasmic synthesis of V1, V2, and V3 polypeptides at 18 h postinfection and subsequent transport to the nucleus by 22 h postinfection. Anti-V1, anti-V2, and anti-V3 sera did not react with intact virions in an immunodiffusion assay, nor did they possess hemagglutination inhibition or viral neutralization activity. Antiserum produced against the four host histone polypeptides (V4 through V7) demonstrated immunofluorescence when reacted with polyoma-infected cells but not with uninfected cells. Antihistone serum was also capable of neutralizing viral infectivity, inhibiting hemagglutination and reacting with whole virions in an immunodiffusion assay.

Animals↗