Effects of Actinomycin D on the Cytopathology Induced by Poliovirus in HEp-2 Cells

One possible mechanism of virus-induced cell damage is that the redistributed (released) lysosomal enzymes produce the cytopathic effect during cytolytic types of infections such as poliovirus in HEp-2 cells. To determine if the lysosomal enzyme redistribution and cell damage are host-cell directed, we studied sensitivity of these events to the action of actinomycin D. By the use of actinomycin D at concentrations producing the least toxicity but maximal effectiveness in shuting down cell RNA synthesis, it was shown that the cytopathic effect and enzyme redistribution were not inhibited and, therefore, not directly controlled and induced by the cell genome in response to the virus infection. Evaluation of cytopathic effect by a phase contrast microscopy method detected changes earlier than the erythrocin B uptake method.     

Phospholipase in the lysosomes of HEp-2 cells and its release during poliovirus infection

A phospholipase was found in the lysosomes of HEp-2 cells. The enzyme exhibited properties typical of other lysosomal enzymes including redistribution from the lysosomes during poliovirus infection. It is proposed that this enzyme may play a role in poliovirus-induced cytopathic effects.     

Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus

Flanagan, John F. (Duke University School of Medicine, Durham. N.C.). Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus. J. Bacteriol. 91:789-797. 1966.-The effect of poliovirus and herpes simplex virus infection on the activity of five hydrolytic enzymes was studied in tissue culture cells of KB type. During the course of poliovirus infection, the activity of beta-glucuronidase, acid protease, acid ribonuclease, acid deoxyribonuclease, and acid phosphatase in the cytoplasm rose to levels two- to fourfold greater than the activity present in the cytoplasm of uninfected cells. The rise in cytoplasmic activity was accompanied by a concomitant decrease in enzymatic activity bound to cell particles. Shift of enzymatic activity from the particulate to soluble state was first detected at 6 hr after poliovirus infection, coinciding with the appearance of new infectious particles and virus cytopathic effect. No net synthesis of these enzymes after poliovirus infection was found. Hydrocortisone added to the culture medium failed to affect either the titer of virus produced in the cells or the release of hydrolytic enzymes from the particulate state. Herpes simplex infection produced minimal alterations in the state of these enzymes in KB cells. It is hypothesized that the breakdown of lysosomes and release of hydrolytic enzymes accompanying poliovirus infection is produced by alterations in cell membrane permeability during the course of virus replication and by the consequent change in the ionic content of the cell sap.     

Tubulin dynamics in cultured mammalian cells

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.     

Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.

Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the level of receptor and within the cell.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

Characteristics of human-mouse hybrid cell lines and the changes induced in them by the poliomyelitis virus

Cells of the four hybrid lines between continuous mouse cells Rag and human diploid embryonal fibroblasts were polymorphic and had mitotic activity in fully formed monolayers. Most of the these mitoses were pathological. Hybrid cells examined 8 months after hybridization were susceptible to the poliomyelitis virus infection with partial cytopathologic effect, they produced virus antigens and the infectious virus. Small hybrid cells displayed a more pronounced cytopathologic effect than did big, polynuclear and mitotic cells. Hybrid cells that were passaged 1.5 months after infection did not excrete any infectious poliovirus but contained poliovirus antigens.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other immunoglobulin-like V-type domains. Analysis of stable cell lines expressing the mutant proteins revealed that virus binding is influenced by mutations in the predicted C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge do not develop cytopathic effect during poliovirus infection, suggesting that poliovirus-induced cytopathic effect may be induced by the virus-receptor interaction.     

Host-Cell Lysosomal Response to Two Strains of Herpes Simplex Virus

A correlation has been made between the host lysosomal responses to and release of infectious virus from HEp-2 cells infected with two strains of herpes simplex virus (HSV). Supravital staining with acridine orange was used for morphological studies of macroplaque and microplaque HSV-infected cells. With the progression of infection, cells infected with either microplaque HSV or macroplaque HSV were observed to undergo different lysosomal and cytopathic changes, which could be correlated with increased accumulation of acid phosphatase and infectious virus in the extracellular fluid.     

Changes in lysosomal enzyme activity in the mitotic cycle of L cells

Activities of lysosomal enzymes (acid phosphatase, N-acetyl-beta-D-glucosaminidase, acid lipase and cathepsin D) have been examined in a synchronized culture of mouse L-fibroblasts. Cell synchronization was achieved by the double thymidine block with a subsequent mitotic selection after colcemid treatment. Specific activities of the enzymes studied were found to be higher in S-G2 that in G1. There is a linear increase (approximate doubling) in enzyme activities per cell from G1 to M. Activity of galactosyltransferase, a marker of the Golgi apparatus, declined in mitotic cells in comparison with the interphase cells. Ultrastructural examination of L-cells revealed a reduction of the intracellular membrane system including the Golgi apparatus during mitosis. Changes in the Golgi apparatus activity have been considered as a possible regulatory point of lysosome formation. The data presented are compared with the results of morphological studies of lysosomal system in L-cells.     

Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

Lysosomal enzymes possess a common antigenic determinant in the cellular slime mold, Dictyostelium discoideum

Antisera have been prepared against two lysosomal enzymes of the cellular slime mold, Dictyostelium discoideum. The two purified enzyme preparations used for immunization, N-acetylglucosaminidase and beta-glucosidase-1, show no cross-contamination with each other and no significant contamination by other lysosomal enzymes. However, antisera raised against either enzyme bind equally well to seven different lysosomal enzymes and show no preference for the enzyme against which they were raised. A total of 10 different antisera have been examined and all show similar results. Preadsorption of antisera with either purified enzyme removes all antibody activity against the other enzyme. Evidence is presented which indicates that the same species of antibodies are responsible for the precipitation of seven lysosomal enzymes. These data are discussed in terms of the proposal that the antigen that is shared by the lysosomal enzymes is a post-translational modification of the enzyme proteins. We have sought to further characterize the distribution of this common antigen among cellular proteins. We show that N-acetylglucosaminidase and beta-glucosidase-1 represent less than 5% of the total common antigen containing proteins in the cell. Precipitation of 35S-labeled cellular proteins from vegetative cells indicates that as much as 15-30% of the total cell protein may possess the common antigen. Preadsorption experiments confirm that all of the proteins immunoprecipitated in these experiments are recognized by the same antibodies that precipitate the lysosomal enzyme activities. Most of the labeled proteins are secreted into the medium along with the lysosomal enzyme activities during axenic growth. During the developmental phase of the life cycle of Dictyostelium, the total amount of the common antigen decreases about 2-fold relative to total cell protein. However, the synthesis of antigenic proteins continues throughout most of development.     

Failure to detect interfering agents in febrile respiratory illnesses

Throat swab specimens from 57 military recruits with febrile respiratory illness in whom no virologic or serologic evidence of infection with respiratory viruses orMycoplasma pneumoniae was found were inoculated and passaged into fresh cultures of monkey kidney cells and continuous lines of human, simian and rabbit cells. Cultures of second passage were challenged with poliovirus type 2 or vaccinia virus and subsequently tested for interference by reading of cytopathic effect and titration of challenge virus. No interference was shown.     

Involvement of the 24-kDa cap-binding protein in regulation of protein synthesis in mitosis

The rate of protein synthesis in metaphase-arrested cells is reduced as compared to interphase cells. The reduction occurs at the translation initiation step. Here, we show that, whereas poliovirus RNA translation is not affected by the mitotic translational block, the translation of vesicular stomatitis virus mRNAs is. In an attempt to elucidate the mechanism by which initiation of protein synthesis is reduced in mitotic cells, we found that the interaction of the mRNA 24-kDa cap-binding protein (CBP) with the mRNA 5' cap structure is reduced in mitotic cell extracts, consistent with their lower translational efficiency. Addition of cap-binding protein complex stimulated the translation of endogenous mRNA in extracts from mitotic but not interphase cells. In addition, we found that the 24-kDa CBP from mitotic cells was metabolically labeled with 32P to a lesser extent than the protein purified from interphase cells. These results are consistent with a hypothesis that the 24-kDa CBP is implicated in the inhibition of protein synthesis in metaphase-arrested cells. Possible mechanisms for this inhibition are offered.     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.