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R A Consigli

Publications and source records attributed to R A Consigli.

At least 55 records · Page 3Linked to original sources
PubMed1986

Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor.

We used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.

Adhesiveness↗
PubMed1986

Two-Dimensional Polyacrylamide Gel Analysis of Plodia interpunctella Granulosis Virus.

The structural polypeptides of purified Plodia interpunctella granulosis virus were analyzed by three different two-dimensional gel systems. Isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of 53 acidic polypeptides in the enveloped nucleocapsid of the virus ranging in molecular weight from 97,300 to 8,000. Nine of these polypeptides were shown to be glycoproteins by the technique of radiolabeled lectin blotting. Separation of the granulin in this system allowed resolution of five species, all of which have identical tryptic peptide maps. This matrix protein was demonstrated to be a phosphoglycoprotein by radiolabeled lectin blotting and acid phosphatase dephosphorylation. Nonequilibrium pH gel electrophoresis followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed resolution of the major basic protein of the virus, VP12, from a more acidic protein of the same molecular weight. Tryptic peptide analysis demonstrated that these two proteins were indeed different and acid urea gels followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed localization of the acidic protein to the envelope and the basic protein to the nucleocapsid of the virus. Finally, probing of the separated envelope nucleocapsid proteins in both the isoelectric focusing and nonequilibrium pH gel electrophoresis two-dimensional systems after transfer to nitrocellulose with iodinated, purified viral proteins allowed further insight into reactions which may be important in the maintenance of the virion structure.

Journal Article↗
PubMed1985

Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells.

A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species.

Animals↗
PubMed1985

Fluorographic detection of [3H]thymidine-labeled deoxyribonucleic acid using agarose gels infused with 2,5-diphenyloxazole prior to electrophoresis.

A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid.

DNA↗
PubMed1985

Glycosylation of purified enveloped nucleocapsids of the granulosis virus infecting Plodia interpunctella as determined by lectin binding.

Using various iodinated plant lectins and a sensitive in vitro lectin binding procedure in which the radiolabeled lectins are applied directly to viral proteins on nitrocellulose sheets transferred from sodium dodecyl sulfate-polyacrylamide gels (Bartles and Hubbard, 1984; Glass et al., 1981), the proteins of the granulosis virus infecting Plodia interpunctella were probed for carbohydrate moieties. Six proteins (Mr 39 700, 31 000, 29 900, 26 300, 17 800 and 12 600) could be detected using a probe for alpha-D-N-acetylgalactosamine. Three proteins were also detectable with the probe specific for alpha-L-fucose (Mr 44 900, 31 000 and 17 800). One protein (Mr 17 800) was detected with the probe for sialic acid.

Animals↗
PubMed1985

Production and characterization of monoclonal antibodies to polyomavirus major capsid protein VP1.

Four hybridoma cell lines producing monoclonal antibodies against intact polyoma virions were produced and characterized. These antibodies were selected for their ability to react with polyoma virions in an enzyme-linked immunosorbent assay. The antibodies immunoprecipitated polyoma virions and specifically recognized the major capsid protein VP1 on an immunoblot. Distinct VP1 isoelectric species were immunoprecipitated from dissociated virion capsomere preparations. Two-dimensional gel electrophoresis demonstrated antibody reactivity with specific VP1 species. Monoclonal antibodies E7 and G9 recognized capsomeres containing VP1 species D, E, and F, while monoclonal antibodies C10 and D3 recognized capsomeres containing species B and C. Two of the monoclonal antibodies, E7 and G9, were capable of neutralizing viral infection and inhibiting hemagglutination. The biological activity of the monoclonal antibodies correlated well with the biological function of the species with which they reacted.

Antibodies, Monoclonal↗
PubMed1984

Isolation and characterization of monopinocytotic vesicles containing polyomavirus from the cytoplasm of infected mouse kidney cells.

Monopinocytotic vesicles containing polyomavirus were isolated from the cytoplasm of mouse kidney cells infected with polyomavirus using sucrose density gradients. Nonenclosed, membrane-associated virions released by the action of neuraminidase separated from vesicle-enclosed virions in the sucrose gradient. Marker enzyme assays indicated the derivation of the vesicle membrane from the plasma membrane of the cell. The 125I-labeled virus enclosed in the vesicle sedimented more slowly in the gradient and was not observed unless infection and endocytosis had occurred. Detergent treatment of virion-containing vesicles caused the release of polyomavirus with sedimentation properties similar to those of purified polyoma virions. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virion proteins from vesicles containing virions demonstrated patterns of proteins similar to those of purified intact virions. Electron microscopy confirmed the presence of single intact virions inside vesicles. The study of these monopinocytotic virion-containing vesicles represents a further step in elucidating the early events of polyomavirus infection.

Animals↗
PubMed1983

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity.

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.

Acetates↗
PubMed1983

Granulosis viruses, with emphasis on the GV of the Indian meal moth, Plodia interpunctella.

The granulosis viruses and nuclear polyhedrosis viruses are being considered for use as biological insecticides for control of their insect hosts. Many of these insect species, which include some of the most serious pests of agriculture and forests, have become difficult to control because they have developed resistance to chemical insecticides. Several laboratory and field studies have demonstrated that the baculoviruses (GV and NPV) are promising alternatives to chemicals for the control of economically important insects. These viruses are highly virulent, selective, and stable, and the impact on the environment following their application is minimal. A decision concerning the application of baculoviruses to stored grain and field crops must be based upon a prudent consideration of the benefits to be obtained and the potential risks of their use. Such decisions should be made only after consideration of the physical, chemical, and biological properties of these viruses. In addition, methods must be developed for the unequivocal identification of these viruses, and their effects on nontarget species at the cellular and molecular levels must be investigated. This can best be accomplished if a sufficient body of knowledge regarding the molecular properties of these viruses and their infection process is accumulated by an extensive quantitative approach. Much of this knowledge is lacking because, prior to their consideration for use as insecticides, the baculoviruses appeared to have little medical or economic importance. As a result, interest in studying them was limited. It has become obvious that the molecular properties of these viruses must be investigated if full advantage is to be taken of using them as insect control agents, and if present and future problems concerning their use as insecticides are to be handled properly. Fundamental research on the biochemical and biophysical properties of baculoviruses has concentrated mainly on a variety of nuclear polyhedrosis viruses (Harrap, 1972a,b; Harrap et al., 1977; Summers and Smith, 1975a,b; Arif and Brown, 1975). Much of this progress can be attributed to tissue culture-host cell systems available for the NPVs. The in vitro host system(s) has allowed insect virologists to make phenomenal strides in understanding the cellular and molecular events of virus infection, and, in addition, to enter the era of biochemical sophistication in which animal virology is found at present.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Generation of capsids from unstable polyoma virions.

Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures.

Capsid↗
PubMed1983

Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region.

As a first step toward identifying the various functional regions of the polyomavirus major capsid protein VP1, we used recently developed methods for the chemical cleavage of proteins and the available polyomavirus sequence data to devise a scheme to produce large, identifiable peptides and generate a cleavage map of VP1. Formic acid (75%) was found to cleave VP1 at only two sites, producing three peptides of apparent molecular weights of 29,000, 16,000, and 2,000. The order of peptides in intact VP1 was determined by recleavage of partial products and was found to be 29,000, 16,000, and 2,000. Two-dimensional peptide mapping studies of 125I-labeled VP1 formic acid peptides established that the limit products of formic acid digestion contained mutually exclusive sets of labeled peptides when either trypsin or chymotrypsin was used and that together the formic acid peptides contained all of the 125I-labeled tryptic and chymotryptic peptides found in VP1. Iodosobenzoic acid (IBA) digestion produced four peptides separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 12,000, 8,000, 7,000, and 5,000. The approximate positions of the IBA peptides in the VP1 sequence were determined by cleavage of formic acid fragments with IBA. The number of peptides produced, their respective sizes, and their order in the intact VP1 molecule agree with predictions made from available sequence data, both for formic acid cleavage and IBA cleavage. In addition, the numbers of 125I-labeled tryptic peptides produced from digestion of VP1 formic acid peptides also agree with predictions made from the sequence information. These data establish with reasonable certainty that the peptides produced by formic acid cleavage and IBA cleavage of VP1 are indeed those predicted. Antibodies raised against spontaneously produced, previously undefined polypeptides resulting from degradation of VP1 reacted exclusively with the formic acid peptides derived from the C-terminal portion of VP1. These antibodies inhibited hemagglutination and neutralized polyomavirus virions. We interpret this to mean that at least some of the antigenic determinants of the receptor moiety reside in this portion of the VP1 sequence.

Amino Acid Sequence↗
PubMed1983

Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region.

The major virion protein of polyomavirus, VP1, consists of about six isoelectric species designated A through F. The minor species D, E, and F are phosphorylated and are thought to serve as viral receptors. We first wanted to distinguish whether all VP1 species are derived by post-translational modification from a common amino acid sequence or whether one or more of the species contain a region(s) of altered amino acid sequence resulting from alternate mRNA processing. We compared the VP1 species by detailed peptide mapping with several combinations of specific protease and radioisotopic labels. This approach enabled us to examine more than 80% of the predicted VP1 sequence, including the amino-and carboxy-termini. We found no evidence of sequence differences among any of the VP1 species. The specific incorporation of 32Pi was found to be the same for all of the phosphorylated species. Comparison of the phosphorylation sites of in vivo 32Pi-labeled D, E, and F by peptide mapping showed them to be identical. Each phosphorylated species contained a single major phosphopeptide and several minor phosphopeptides. The major phosphoamino acid, identified by acid hydrolysis, was phosphothreonine, with phosphoserine also present. By using chemical cleavage methods, we localized the major phosphorylation region to a central portion of the VP1 sequence. We discuss some features of this region and relate this information to functional implications of phosphorylation.

Amino Acid Sequence↗
PubMed1982

Detection and quantitation of Newcastle disease virus proteins in infected chicken embryo cells.

A technique was analyzed by which Newcastle disease virus (NDV) proteins could be quantitatively detected in the presence of chicken embryo cellular proteins in NDV-infected cells. The technique involved removal of electropho-proteins from a sodium dodecyl sulfate-polyacrylamide-agarose gel matrix by chemical cleavage of the acrylamide gel cross-linker. The proteins were subsequently transferred and covalently bound to diazobenzyloxymethyl paper. By incubating the paper with unlabeled antisera and 125I-labeled Staphylococcus aureus protein A, the specificity of the antisera and the sensitivity of this method of quantitative antigen detection were tested. The results demonstrated that as little as 1 ng of an individual NDV protein could be detected. Furthermore, this technique can simultaneously quantitate the synthesis of multiple NDV proteins under experimental conditions in which immunofluorescence, hemadsorption, and plaque assays failed to show virus protein synthesis or the formation of virus progeny.

Animals↗
PubMed1982

Improved infectivity of reassembled polyoma virus.

Polyoma virus was dissociated into capsomeres (18, 12, and 5S) and a DNA-protein complex (48S) with the Ca2+ chelator, ethyleneglycol-bis-N,N'-tetraacetic acid, and the reducing agent, 2-mercaptoethanol. The reaction was maintained at pH 5.0. Reassembly of the dissociated components to complete virions was accomplished by dialyzing these components overnight at 4 degrees C against the reassembly buffer containing CaCl2, dimethylsulfoxide, Triton X-100, and 0.01 M Tris-acetic acid (pH 5.0). Reconstructed particles ranged from 240S complete virions to lighter intermediate species. Approximately 25% of the dissociated particles could be physically reassembled to complete virions. These virions regained 12.5% of their hemagglutination ability and as much as 6.7% of their original infectivity. The infectivity of these reassembled particles represented a 100-fold increase in infectivity compared with that of the particles that were dissociated and reassembled at pH 7.4. Biochemical analysis showed that the polyoma viral receptor of the virions reassembled at pH 7.4 was greatly reduced, whereas virions reassembled at pH 5.0 retained their receptor. Reassembly could be further improved by additions of either exogenous capsomeres or DNA-protein complex to the reassembly reaction mixture.

Calcium Chloride↗