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Biomedical subjects

R A Consigli

Publications and source records attributed to R A Consigli.

At least 91 records · Page 5Linked to original sources
PubMed1977

Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions.

Analysis of polyoma virions by X-ray fluorometry demonstrated that calcium (Ca2+) was associated with the purified virion. Treatment of purified virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, and the reducing agent dithiothreitol caused the virions to dissociate. Electron microscopy revealed that the virions were dissociated to the capsomere level. Incubation of polyoma virions with 150 mM NaCl, 10 mM EGTA, and 3 mM dithiothreitol was optimum for the dissociation reaction. The pH for the dissociation reaction ranged from 7.5 to 10.5. Cesium chloride density gradient centrifugation indicated that both EGTA and dithiothreitol were necessary for dissociation to occur; neither reagent alone dissociated the virus. The major protein product of the dissociated viral particles sedimented at 12S. Relationships between these experiments and the alkaline carbonate-bicarbonate dissociation of polyoma are discussed.

Calcium↗
PubMed1976

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells.

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.

Cell Line↗
PubMed1976

Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus.

A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus. The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein. The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl. The isolated protein can bind to both circular and linear duplex DNA. Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients. The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template. The binding protein itself is free of detectable DNA polymerase or nuclease activity.

Binding Sites↗
PubMed1976

Early events in polyoma virus infection: attachment, penetration, and nuclear entry.

The plaque-assay technique was used as a tool to determine the optimal conditions for adsorption of polyoma virions to host cells. Using these optimal conditions of adsorption, an electron microscopy study of the early events of infection was performed. By electron microscopy and autoradiography, it was demonstrated that both the viral coat proteins and DNA arrive simultaneously in the nucleus as early as 15 min postinfection. When horseradish peroxidase-labeled virions, pseudovirions, and capsids were used to infect cells, only the particles with nucleic acid or a factor(s) associated with the nucleic acid, i.e., histones, appeared to enter the nucleus. Moreover, when virions were used to infect either permissive or nonpermissive cells, identical early events of viral infection, i.e., adsorption, penetration, and nuclear transport, were observed, suggesting that these early events of infection are a property of the virion and not the host cell.

Adsorption↗
PubMed1976

Early events in the lytic infection of primary mouse kidney cell culture with polyoma virus. The effect of various input multiplicities.

Early polyoma (Py) virus-specific RNA synthesis was examined in cells infected with different concentrations of Py-virus. The effect of various multiplicities of infection (m.o.i.) on the rate of Py-RNA synthesis is different at 30 hr as compared to 65 hr. Thirty hours after infection at 27degrees, in the presence of 5-fluoro-2-deoxyuridine (FdU), an increase in input multiplicity was not associated with a quantitatively commensurate increase in the amount of virus-specific RNA synthesized. At 65 hr, the amount of viral RNA synthesized was roughly proportional to the number of infecting virus particles.

Cells, Cultured↗
PubMed1975

Origin of the polyoma virus-associated endonuclease.

Endonuclease activity can be found associated with highly purified preparations of polyoma virus. Evidence has been obtained that this enzyme is not an integral part of the virus but is contributed by the fetal calf serum used in maintenance of polyoma-infected cells. This finding is based on: (i) the lack of virion-associated endonuclease activity when virus is produced using serum-free media and (ii) the production of polyoma antibody which neutralizes fetal calf serum endonuclease activity.

Animals↗
PubMed1974

Effect of beta-propiolactone inactivation of polyoma virus on viral functions.

Polyoma virus was inactivated by treatment with beta-propiolactone. T-antigen production, polyoma-RNA synthesis, induction of host DNA synthesis (measured by incorporation of labeled thymidine into the cell culture), and in vitro transforming ability were inactivated to a similar degree by various beta-propiolactone concentrations (0.25% beta-propiolactone reduced these functions approximately 96%), whereas plaque-forming ability and the ability of the virus to replicate its DNA and to synthesize capsid antigen were inactivated by a given concentration of beta-propiolactone to a much greater degree (0.25% beta-propiolactone led to a reduction of plaque-forming ability of over 8 logs). The significance of these data and their relationship to previously published experiments are discussed.

Animals↗
PubMed1974

Characterization of polyoma DNA-protein complexes. I. Electrophoretic identification of the proteins in a nucleoprotein complex isolated from polyoma-infected cells.

A study was undertaken to examine polyoma DNA-protein complexes. A biophysical characterization of the complexes was made, and the proteins found in such complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison was made between a 52S nucleoprotein complex isolated from nuclei of 26-h polyoma-infected cells and a 28S virion core complex ejected out of mature virus particles. It was found that both complexes were reduced to a 20S viral DNA component plus free protein after incubation in 1 M NaCl or Sarkosyl. Treatment of the complexes with either Pronase or 0.5 M NaCl resulted in only partial removal of proteins from the viral DNA. After fixation in formaldehyde, the 52S nucleoprotein complex had a buoyant density of 1.45 g/cm(3), and the virion core complex had a buoyant density of 1.59 g/cm(3). Sodium dodecyl sulfate-polyacrylamide gel profiles of purified polyoma virion proteins, used as a reference marker, demonstrated three capsid proteins, V1 to V3, as well as four histones, V4 to V7, which constituted about 7% of the total virion protein. Electrophoretic analysis of the proteins comprising the 52S nucleoprotein complex revealed that the same seven proteins present in the mature virion were also found in this complex. However, the ratios of the proteins in the complex were quite different from that of the mature virion, with the four histones comprising 48% of the total complex protein. A pulse-chase experiment of the nucleoprotein complex demonstrated that the 26-h complex was chased into mature virions.

Animals↗
PubMed1974

In vitro radioisotopic labeling of proteins associated with purified polyoma virions.

An in vitro procedure using [(14)C]formaldehyde and sodium borohydride was used to label the proteins of purified polyoma virions. This method, which labeled the three capsid proteins and the four internal histones of polyoma virus, did not alter the biological and biophysical characteristics of the virus. In addition to the seven viral proteins, five additional radioactive peaks were resolved on polyacrylamide gels. Four of these peaks could be attributed to growing the virus in the presence of serum. The fifth peak appears to be a nonhistone host-contributed protein made before infection.

Carbon Radioisotopes↗
PubMed1972

Transient inhibition of polyoma virus synthesis by Sendai virus (parainfluenza I). I. Demonstration and nature of the inhibition by inactivated virus.

Superinfection of polyoma virus-infected mouse embryo cells by beta-propiolactone-inactivated Sendai virus resulted in a 90% inhibition of the synthesis of infectious polyoma progeny. The interference is dependent upon the time of superinfection and the concentration of the inactivated virus. The inhibition of polyoma virus synthesis is transient in nature since normal synthesis of polyoma progeny virus is seen upon prolonged incubation. Interferon does not appear to be implicated in the interference. Various aspects of the biological and synthetic capabilities of beta-propiolactone-inactivated Sendai virus are also described.

Animals↗