Biomedical subjects

E Everitt

Publications and source records attributed to E Everitt.

At least 19 recordsLinked to original sources

PubMed2000

Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigmaC.

Avian reovirus protein sigmaC, the viral cell-attachment protein, is a minor component of the outer-capsid shell of the viral particle that is synthesized in small amounts in infected cells. We cloned the sigmaC-encoding ORF in vector pIL-2f, expressed it in Escherichia coli, and partially purified the resulting recombinant protein from inclusion bodies. Rabbit polyclonal antibodies raised against the recombinant protein specifically recognized the viral polypeptide in ELISA, immunoprecipitation, and Western blotting. To study the oligomerization capacity and cell-binding affinity of protein sigmaC, the sigmaC-encoding ORF was also expressed in chicken embryo fibroblasts (CEFs) and in reticulocyte lysates. In all three systems protein sigmaC is expressed as a multimer with identical electrophoretic mobility to the naturally occurring protein. Cell-binding experiments show that both in vitro and in vivo expressed protein sigmaC display affinity for CEF receptors, and this property is exclusively associated with the oligomeric form of the protein. The fact that incubation of CEF cells with the recombinant protein expressed in bacterial cells completely blocks the binding of purified reovirions indicates both that binding of this protein to cells is specific and saturable, and that reovirions and protein sigmaC bind to the same class of cell receptor. Saturation binding experiments, performed with the recombinant protein expressed in E. coli and with purified reovirions, showed that the number of cellular receptor sites (CRSs) for avian reovirus S1133 is 1.8 x 10(4) per CEF cell, whereas the number of cellular receptor units (CRUs) for sigmaC is 2.2 x 10(5) per CEF cell. These results are consistent with previous reports on the binding of mammalian reoviruses.

Animals↗

PubMed2000

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins.

The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.

Animals↗

PubMed1999

Adenovirus cellular receptor site recirculation of HeLa cells upon receptor-mediated endocytosis is not low pH-dependent.

HeLa cells were depleted of 75% of the available adenovirus type 2 specific cellular receptor sites (CRSs) by controlled attachment of viruses at a high multiplicity of infection. Upon removal of unattached viruses and further incubation, the cells were able to recycle the CRSs to 75% of the level of uninfected control cells within 5 h. The rate of virus receptor recycling was approx. 1 CRS x min-1 x cell-1. The existence of receptor recycling further strengthens the involvement of a total process of receptor-mediated endocytosis in adenovirus internalization. The recirculation process was neither affected by the lysosomotropic agents ammonium chloride and amantadine-HCl, nor by the ionophore monensin or the multifunctional weak-base amine chloroquine.

Adenoviruses, Human↗

PubMed1999

Identification of IPNV-specified components released from productively infected RTG-2 cells following massive cytopathic effect.

Rainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

Animals↗

PubMed1999

Synthesis of a eukaryotic virus protein in a prokaryotic viral-cell system: production of the adenovirus type 2 fiber shaft fragment by a tightly regulated T7POL-M13 expression system.

The use of recombinant technology for the production of proteins of interest in biotechnology and medicine has grown immensely during the last decade. A major problem often encountered is the degradation of the recombinant product by host cell proteases. We developed a novel system based on the cloning and expression of an inducible phage T7 RNA polymerase into the main intergenic region of the phage M13-KO7. After infection of permissive bacterial strains with the engineered phage, the polymerase gene is transcribed, subsequently translated and gene fragments cloned under T7 promoter sequences are then transcribed. For the evaluation of this system, the gene encoding the shaft fragment of the adenovirus type 2 fiber was cloned into a pET 3a-based expression vector. Expression was demonstrated in a BL21(DE3) strain (containing one copy of the T7 RNA polymerase gene) and also in several F pili-containing bacterial strains only after infection with the proper bacteriophage. Several important parameters for heterologous gene expression in Escherichia coli were investigated. Different bacterial strains were evaluated for the production of the recombinant protein, following: the expression levels, the growth rates and the stability of the plasmid vector at different time intervals after induction. It was observed that the expression levels as well as division rates and plasmid stability differed between the different bacterial strains. The best expression levels were obtained when using the E. coli Top IOF' strain. Degradation was only observed in BL21(DE3) cells after 6 h of induction, whereas none of the F'-containing cells were shown to degrade the recombinant protein during the time of expression. This system, based on the T7 pol-M13 bacteriophage, was shown to be very tightly regulated for most of the bacterial strains evaluated with no expression before induction of the T7 RNA polymerase.

Adenoviruses, Human↗

PubMed1999

TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus.

Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4-8 degrees C for 2 months in the salt buffer used commonly.

Animals↗

PubMed1999

Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2.

Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline beta elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.

Animals↗

PubMed1996

Adenovirus uncoating and nuclear establishment are not affected by weak base amines.

We have used four established lysosomotropic agents, ammonium chloride, amantadine, chloroquine, and methylamine, to monitor the possible interference with an early low-pH-dependent step during adenovirus replication. Two concentrations of each of the different agents were selected; one was essentially nontoxic to uninfected HeLa cells, and the other resulted in some toxicity as measured by trypan blue staining and by interference with cell monolayer establishment, cell proliferation, and radioisotope labelling. It was separately determined that these concentrations displayed pH-raising effects of the same magnitude as higher concentrations previously used in similar studies. Adenovirus uncoating in vivo, normally reaching its maximum within 1 h after infection, was not affected by any of the agents. The subsequent levels of successful nuclear entry events by the parental genomes were monitored by measuring the extent of transcription of an mRNA species coding for the early 72-kDa DNA-binding protein at 10 to 12 h postinfection. In HeLa, KB, HEp-2, and A549 cells, none of the agents were able to affect the levels of early transcription after administration at the point of infection or at 3 h after infection. The cumulative synthesis of the hexon antigen was assessed late in infection, and inhibitory effects were revealed upon administration of 10, 20, and 40 mM ammonium chloride, 10 mM methylamine, and 0.5 mM amantadine, irrespective of the time point of addition. Ammonium chloride at 5 mM reduced the hexon yield by 20% at the most when added within 50 min after infection. Chloroquine at concentrations of 2.5 and 5 microM specifically reduced the hexon yields by 30 to 40% when administered within the first 50 min of infection. On the basis of the lack of effects of nontoxic concentrations of the four agents on the early virus-cell interactive event of uncoating and the early virus-specified transcription, we conclude that a low-pH-dependent step early in the adenovirus replication cycle is not mandatory for a successful infection.

Adenoviridae↗

PubMed1994

Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific infectivity.

An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described. Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1. IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl. The bovine serum albumin content of the growth medium supplement also elutes close to this position. Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).

Animals↗

PubMed1993

Enhancement of intracellular uncoating of adenovirus in HeLa cells in the presence of benzyl alcohol as a membrane fluidizer.

The early extra- and intra-cellular interaction between adenovirus type 2 and HeLa cells was studied in the presence of benzyl alcohol as a fluidizing agent. The process of virus attachment and internalization were not affected at 5-15 mM of benzyl alcohol at 25 degrees C. Under the same conditions an enhancement by 45% at the most was demonstrated for the cell-mediated virion uncoating. By completely blocking virion internalization with 50 mM azide the uncoating was reduced to 20% of the normal level. The remaining surface-located uncoating was not affected by benzyl alcohol. It was demonstrated that an enhancement of the intracellular virion uncoating was followed by a raised production of the hexon antigen, which was interpreted as an increase in the specific infectivity of the virus.

Adenoviridae↗

PubMed1992

Antibody-mediated uncoating of adenovirus in vitro.

In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.

Adenoviruses, Human↗

PubMed1991

Infectious entry pathway of adenovirus type 2.

Internalization of the infectious fraction of human adenovirus type 2 into HeLa cells was followed by a quantitative internalization assay. Treatments known to selectively block receptor-mediated endocytosis reduced the internalization of infectious virus to an extent close to the reduction of endocytosis of transferrin. This suggests that one of the first steps in the infectious cycle of adenovirus type 2 is internalization by the coated-pit and -vesicle pathway.

Adenoviruses, Human↗

PubMed1990

Estramustine phosphate reversibly inhibits an early stage during adenovirus replication.

Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

Adenoviridae Infections↗

PubMed1990

Cultivation of HeLa cells with fetal bovine serum or Ultroser G: effects on the plasma membrane constitution.

Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G- supplemented cells, respectively. Plasma membranes from cells grown with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells. The former membranes also showed a 3 times higher specific [3H]acetate labeling of cholesterol, indicating a higher de novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe.

Blood↗

PubMed1990

Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction.

Adenoviruses, Human↗

PubMed1987

Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained cellular protein.

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.

Amido Black↗

PubMed1985

Assessment of specific virus infectivity and virus neutralization by a progeny virus immunotitration method as exemplified in an adenovirus system.

An alternative method for the determination of specific virus infectivity and quantitative measurement of inhibitory activity of antibodies was developed using an adenovirus system. HeLa cells in 37 ml suspension cultures of 1.5 X 10(7) cells were infected with purified adenovirus 2 (Ad2) at different multiplicities of infection. After appropriate incubations, total progeny virus was isolated by a one-step CsCl gradient sedimentation procedure. Recovered virions were disrupted in the presence of 5 M urea and directly quantitated by rocket immunoelectrophoresis against an anti-hexon-antiserum. One infectious unit (IU) was defined as the lowest amount of virions capable of producing maximum yield of progeny virus in the standardized system, and corresponded to 32 physical particles, which also equalled one plaque-forming unit (pfu). The coefficient of the inter-experimental variation of the total virus yield determination was 13%. Reduction in progeny virus synthesis was taken as a measurement of the degree of the inhibitory effect of neutralizing antibodies. A linear relationship was obtained between dilution of a neutralizing antiserum and reduction in synthesis of progeny virus. Separate determinations of such neutralization revealed a coefficient of variation of 5.5%.

Adenoviruses, Human↗

PubMed1985

Human adenovirus 2 as immunogen in rabbits yields antisera with high titers of antibodies against the nonstructural 72K DNA-binding protein.

Immunization in rabbits with intact, highly purified adenovirus type 2 (Ad2) virions, yielded antisera with high titers of antibodies against the 72 000 dalton DNA-binding protein (DBP). This was established by rocket immunoelectrophoresis when an anti-intact Ad2-antiserum was analyzed against fractions from an ion-exchange chromatogram of soluble antigens remaining after virus isolation from virus-infected HeLa cells. The high anti-DBP titer did not reflect the composition of the immunogen, since no DBP was detectable within virions. An antiserum raised in response to mildly disrupted virions showed no specificity against the DBP, but contained antibodies against the same structural proteins as the anti-intact Ad2-antiserum, when analyzed by the immunoblotting technique. These findings indicate that the nonpermissive rabbit as an experimental host permits early gene expression of a human adenovirus.

Adenoviruses, Human↗