Virion glycosylation governs integrity and infectivity of infectious pancreatic necrosis virus.

The possible importance of the O-linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O-glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re-isolated after rate-zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re-isolation of viruses, a 3-5 (10)log-unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re-evaluation of reading values from UV-spectra of purified IPNV yielded a specific infectivity of 3 × 10(11) TCID(50)-units mg(-1) of protein and a ratio of 40 virions per TCID(50)-unit in the CHSE-214 cell system.

SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2-dependent binding to the extracellular matrix.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a Ca+2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells. Bovine aortic endothelial cells, exposed to 2-40 micrograms SPARC/ml per 2 x 10(6) cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions. SPARC bound Ca+2 in both amino- and carboxyl-terminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca+2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca+2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.

Fibronectin enhances the migration rate of human neutrophils in vitro.

Efficient polymorphonuclear neutrophil (PMN) migration depends on specific interactions between PMNs, endothelial cells, and extracellular matrix (ECM) proteins. We investigated the relationship between PMN migration and the ECM molecule fibronectin (FN). We used an in vitro migration assay system to show that human PMNs migrated across an FN-coated filter barrier toward a formyl-Met-Leu-Phe (fMLP) chemoattractant gradient in greater numbers than across (uncoated) bare fitters. In 1 h of fMLP stimulation, 69 +/- 6% of the PMNs had migrated across the FN-coated filters, whereas 46 +/- 5% of PMNs migrated across bare filters. This effect was specific to FN; coating the filters with the ECM protein vitronectin did not enhance migration. Monoclonal antibodies against FN or against the alpha5 or beta1 integrin subunits of the FN receptor inhibited the enhanced PMN migration response across FN-coated filters. These findings indicate that the extracellular matrix protein FN enhances PMN migration and that this response is mediated by the alpha5beta1 FN receptor.

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.

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.

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%.

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.

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).

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.

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.

Quantitation of protein by alkaline extraction of naphthol blue black-stained polypeptides in sodium dodecyl sulfate-polyacrylamide slab-gels.

A sodium dodecyl sulfate-polyacrylamide slab-gel system was used to study the alkaline extraction of Naphthol Blue Black B-stained polypeptides. A simple, rapid, and reproducible method for quantitative assessment of 1- to 5-micrograms quantities of individual polypeptides in the molecular weight range of 14,000 to 120,000 is described. The working range of the assay may be extended to the 20-micrograms level at the expense of rapidity.

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.

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.

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.

Virus receptor interaction in the adenovirus system. II. Capping and cooperative binding of virions on HeLa cells.

Adenovirus type 2 attachment to HeLa cells was analyzed under controlled conditions. The temperature-dependent attachment kinetics revealed an inflection point at around 20 degrees C, and above this temperature the increase of the rate was doubled. In multiplicity dependence experiments, the attachment exhibited positive cooperative binding at 37 degrees C. This binding pattern was inhibited by low temperatures and prefixation of cells with 0.015% glutaraldehyde. Attachment of rhodamine-labeled virions revealed capping of the particles on 15% of the cells at 37 degrees C. Capping was inhibited by low temperatures, glutaraldehyde fixation of cells, and treatment with cytochalasin B, azide, and 2-deoxyglucose. Consequently, we propose that the adenovirus type 2 attachment to cells leads to rearrangements in the plasma membrane, resulting in cooperative binding and capping of the virus particles.

Expression of SPARC is correlated with altered morphologies in transfected F9 embryonal carcinoma cells.

SPARC (secreted protein, acidic and rich in cysteine) is a Ca(2+)-binding glycoprotein that has recently been identified as a member of a group of proteins that exert antispreading effects on various cultured cells. In addition, SPARC is induced during the later stages of F9 stem cell differentiation to parietal endoderm (PE). When treated with retinoic acid and dibutyryl cAMP, F9 cells differentiate into PE and SPARC mRNA is increased approximately 20-fold. To determine whether the chronic overexpression or inhibition of expression of SPARC would affect the morphology, attachment, or differentiation of F9 cells, we transfected undifferentiated F9 cells with cDNA encoding SPARC or anti-sense SPARC and cloned lines that expressed either elevated or reduced levels of SPARC protein. The transfected F9 cells displayed altered morphologies in culture: cells of four overexpressing lines appeared clumped and rounded, whereas those of three underexpressing lines were spread and flat, in comparison to controls. Moreover, the morphological differences persisted during differentiation of the lines to PE. The altered morphology was not due to an increased expression of collagenases and did not affect the ability of the cells to attach and adhere to tissue culture plastic. The altered phenotype of the transfected F9 cells appeared to be directly related to the level of extracellular SPARC. Since overexpression of SPARC induced rounding and aggregation of F9 cells in culture, we propose that SPARC facilitates modulation of cell-cell or cell-substrate interactions in vivo.

Overexpression of SPARC in stably transfected F9 cells mediates attachment and spreading in Ca(2+)-deficient medium.

The Ca(2+)-binding protein SPARC is one of a group of proteins that function in vitro to promote the rounding of cells. To assess whether the modulation of cell shape by SPARC is affected by extracellular Ca2+, we used F9 cell lines that had been stably transfected with sense or antisense SPARC DNA. Sense-transfected (S) lines that overexpress SPARC are aggregated and rounded, whereas antisense (AS) lines that express low levels of the protein are flat and spread. We tested whether the cell lines would exhibit these altered morphologies in Ca(2+)-deficient media. When cultured under these conditions, S lines attached and spread, whereas AS lines attached but remained round, with no subsequent spreading. Addition of CaCl2 or purified SPARC to the Ca(2+)-deficient medium resulted in spreading of the AS and control lines and a reappearance of the altered morphologies. Expression of the Ca(2+)-binding cadherin uvomorulin by the cell lines correlated with neither their morphology nor their level of SPARC expression. We conclude that the altered phenotypes of the transected lines reflect, in part, the concentration of extracellular Ca2+ and that the spreading exhibited by the S lines under Ca(2+)-deficient conditions is directly related to their enhanced expression of SPARC. SPARC might, therefore, mediate interactions between cells and matrix that are permissive for adhesion when levels of extracellular Ca2+ are diminished.

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.

A capture enzyme-linked immunosorbent assay for virus infectivity titrations as exemplified in an adenovirus system.

An enzyme-linked immunosorbent assay (ELISA), employing a capturing antihexon monoclonal antibody specifically recognizing free hexons, was developed for quantitative infectivity titration of adenovirus in a microscale titration assay. The method is based on the quantitative assessment of the total excess production of the major structural protein late in infection in samples consisting of 10(5) virus-infected HeLa cells maintained as stationary suspension cultures. Results are obtained with a coefficient of variation of 10% within 50 hours after virus infection. The method was designed for monitoring substances interfering with viral replication, e.g., neutralizing antibodies or antiviral drugs. Since it measured the total antigen content associated with cells as well as antigens possibly released into the growth medium the general approach should be applicable to any viral system where a structural protein is synthesized in excess.

Mechanism of the arginine requirement for adenovirus synthesis. I. Synthesis of structural proteins.

The mechanism of the arginine requirement for adenovirus was studied in cultures of KB cells infected with adenovirus type 2. Macromolecular synthesis was found to be severely impaired in uninfected cells under complete arginine deprivation, whereas an arginine concentration of 50 mum yielded a moderate and reversible inhibition of growth and nucleic acid synthesis. At this concentration, viral structural proteins were accumulated in excess although the virus yield was reduced more than 1,000-fold. The arginine-sensitive step appeared to occur early during the first 15 hr postinfection in the virus growth cycle. Virus-infected cells deprived of arginine to 50 mum showed, when reversed, a 4- to 5-hr lag period before the increase in virus growth was observed. Analysis of the radioactive pattern of labeled virions synthesized after reversion showed that all polypeptides were synthesized after addition of arginine to the medium, and none of the virion-polypeptides which are revealed by gel electrophoresis appeared to be preferentially synthesized after arginine reversion. The excess pool of structural proteins formed during depletion appeared to a large extent to be unavailable for virus assembly.