Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.     

In vitro treatment of HEp-2 cells with human tumor necrosis factor-alpha and human interferons reduces invasiveness of Salmonella typhimurium

Enteroinvasive bacteria, like Salmonella typhimurium, can be internalized in in vitro cultured epithelioidal cells, like HEp-2 cells. This phenomenon is inhibited by pretreatment of cells with human tumor necrosis factor alpha (TNF-alpha) in a dose- and time-dependent manner. The effect was also reproduced in other cell types, including diploid embryo fibroblast cells. The TNF-alpha effect was neutralized by anti-TNF-alpha antibodies. No synergistic effect was produced by combinations of TNF-alpha with either interferon alpha or gamma. Unlike the effects of interferons, TNF-alpha inhibited the invasiveness of Shigella flexneri and the TNF-alpha effect was not inhibited by cycloheximide.     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Differential cell membrane labilizing effect of adenovirus type 5 and 12 observed during productive and abortive infection.

Culture fluid of human epitheloid (HEp-2) cells was examined for extracellular lactate dehydrogenase activity as an indicator of cell damage during a 48 h period in which virus replication and changes in cell morphology occurred. Uninfected and adenovirus type 5-infected cells had the same levels of extracellular enzyme activity both before and after the appearance of morphological changes in cells due to virus infection, whereas adenovirus type 12-infected cells showed increased extracellular enzyme activity. Cells infected with either adenovirus type 5 or type 12 had the same total cellular and extracellular lactate dehydrogenase activity. Hydrocortisone, a membrane stabilizing agent, prevented abnormal leakage of lactate dehydrogenase from adenovirus type 12-infected cells, but had no effect on virus replication or total enzyme activity of infected cells. After inoculation of monkey kidney (Vero) cells the yield of progeny adenovirus type 5 virions was greatly reduced and there was no production of adenovirus type 12 virions. The pattern of extracellular lactate dehydrogenase activity of uninfected and adenovirus type 5- and type 12-infected Vero cells was like that with HEp-2 cells. Therefore, production of adenovirus type 12 virions is not necessary for the virus-cell interaction causing cell membrane labilization.     

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.     

Variation of Interferon Production During the Cell Cycle

The capacity of cells to produce interferon has been found to depend on the phase in the cell cycle at which virus infection took place. Monolayer cultures of L cells were synchronized by the double thymidine-block method. Such synchronously growing cultures were used to study the ability of cells to produce interferon when they were infected with ultraviolet-inactivated Newcastle disease virus (UV-NDV) at different phases of the cell cycle. In all instances, interferon was detected early and reached a maximum at about 16 hr after infection. However, the levels of interferon found in medium of cultures infected at early post-deoxyribonucleic acid (DNA) synthetic (G2) and to some extent at late G2 phases of the cell cycle were comparatively lower than those found in cultures infected at the early DNA synthetic (S) phase. There appeared also in these infected growing cultures a transient period when interferon production was apparently delayed. This period corresponded interestingly with the time of mitotic burst. Infection of thymidine- or 1-beta-d-arabino-furanosylcytosine-inhibited cultures with UV-NDV also led to similar interferon response as that observed in growing cultures infected at early S. However, no transient delay of interferon production was demonstrated in these cultures.     

Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures

Hallum, J. V. (University of Pittsburgh, Pittsburgh, Pa.), and J. S. Youngner. Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacteriol. 92:1047-1050. 1966.-The effect of interferon on single cycles of replication of vesicular stomatitis virus and Mahoney poliovirus ribonucleic acid was studied in chick embryo cell cultures. The results showed that the titer of a given interferon preparation was independent of the input multiplicity of the challenge virus. In addition, the increase in virus yield with increasing virus challenge was a function of the number of infected cells, each of which yielded progeny at a level determined by the concentration of interferon to which the cells were exposed. These findings are not compatible with the concept that increases in the size of the virus challenge reverse or overcome protection of cells by interferon.     

Effect of lactoferrin on enteroaggregative E. coli (EAEC).

We previously demonstrated that lactoferrin inhibits adherence of enteropathogenic Escherichia coli to HEp-2 cells and decreases invasiveness of Shigella flexneri in HeLa cells by disruption of the type III secretory system (TTSS) of both enteropathogens. To determine whether these effects were specific to the TTSS, we assessed the activity of bovine lactoferrin on enteroaggregative E. coli (EAEC), enteropathogens whose virulence is not TTSS dependent. Bovine lactoferrin at a concentration of 1.0 and 0.1 mg/mL inhibited EAEC growth. Saturation with iron reversed the bacteriostatic effect. Lactoferrin under nonbacteriostatic conditions decreased EAEC adherence to HEp-2 cells as evaluated by microscopy and CFUs; this effect was not iron dependent. Lactoferrin inhibited EAEC biofilm formation and increased autoagglutination. Lactoferrin blocks EAEC adherence by inducing release and degradation of aggregative adherence fimbria, a key element of EAEC pathogenesis. We hypothesized that lactoferrin binding to lipid A of lipopolysaccharide disrupts the virulence proteins anchored to the bacterial outermembrane. These data suggest that the effect of lactoferrin on surface proteins is not restricted to organisms having a TTSS.     

Effect on drugs changing the intracellular level of adenosine 3',5'-cyclic monophosphate on interferon formation in chick embryo cells of different ages]

The effect of theophylline and adrenaline on the synthesis of interferon induced by the influenza B virus, strain Lee, in a chick embryo tissue culture was studied. Both preparation were found to decrease interferon synthesis when 5-day-old cultures were used; the inhibitory effect was increased when the two drugs were used together. The degree of inhibition of interferon production depended on a dose of the preparation; the inhibition was still present even when the drugs ere introduced several hours after the cells were infected with interferonogen. The treatment of one-day-old cultures with theophylline resulted in increase of interferon synthesis, whereas administration of adrenaline alone or together with theophylline did not affect the level of interferon synthesis. The drugs used produced no effect on the reproduction of the test-virus of vesicular stomatitis, Newcastle disease and Chickungunya viruses in chick embryo cells and influenza B virus in the developing chick embryos. The results obtained are discussed from the point of view of a possible influence of the intracellular adenosine 3',5-cyclic monophosphate level on the synthesis of virus-induced interferon.     

Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein sigmaA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein sigmaA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Identification of functional binding sites for progesterone in rat Leydig cell plasma membrane.

Steroid hormones influence cell functions by binding to intracellular receptors and then acting within the nucleus. There is now evidence that steroids affect cell functions also via interaction with plasma membrane receptors in a number of different cell types. In this regard, progesterone appears to be one of the most active steroids. In this paper, we evaluate the effects of progesterone on rat Leydig cell functions, determining variations of ion homeostasis and testosterone production. This steroid was able to effect a depolarization of the plasma membrane that was due to an influx of sodium (Na+) from the external medium since it was absent when extracellular Na+ was iso-osmotically substituted with choline chloride or sucrose. The determination of intracellular sodium concentration ([Na+]i) with the Na+ -sensitive fluorescent dye sodium-benzofuran-isophtalate (SBFI) confirmed these observations. Progesterone did not modify Leydig cell intracellular calcium concentration ([Ca2+]i) at any dose tested. Furthermore, using a cell impermeant progesterone conjugate, we demonstrated that progesterone was able to stimulate Leydig cell steroidogenesis in a dose-dependent manner. The exclusion of calcium (Ca2+) from the extracellular medium did not modify the depolarizing action of progesterone and its steroidogenetic effect while in Na+ -free medium (sucrose supplemented) progesterone-stimulated effects were completely blunted. Finally, using fluorescence microscopy with a fluorescein isothiocyanate-coupled cell impermeant progesterone conjugate, we identified plasma membrane binding sites for progesterone in rat Leydig cells. These results suggest that rat Leydig cells possess progesterone receptors located on the plasma membrane, which when occupied achieves a plasma membrane depolarization, dependent on an influx of Na+ from the external medium, and the subsequent activation of steroidogenesis.     

Inhibition of Na transport by 2-chloroadenosine: dissociation from production of cyclic nucleotides

The adenosine analogue 2-chloroadenosine (2-CA) is often used to determine the biologic effects of adenosine because 2-CA is less susceptible to degradation than adenosine. We studied the effects of 2-CA on primary cultures of rat inner medullary collecting ducts because there is good evidence that adenosine can influence cell function through its effects on second messengers. 2-CA inhibited Na+ transport across the apical membrane and increased cAMP content of the cells. The major adenosine receptors in these cells appear to be the stimulatory (A2) type. Stimulation of cAMP by 2-CA was more potent when applied to the apical membrane than to the basolateral membrane, an effect opposite to that of vasopressin. These results imply that adenosine receptors are more numerous or more effective on the apical membrane than on the basolateral membrane. Inhibition of Na+ transport was probably not mediated by an adenosine receptor as evidenced by (i) a lack of effect of adenosine and other adenosine analogues on Na+ transport; (ii) a lack of effect of nonmetabolizable cyclic nucleotides on Na+ transport; and (iii) a clear discrepancy in the temporal course of 2-CA effects on a second messenger system (cAMP) and 2-CA inhibition of Na+ transport. Dipyridimole, an inhibitor of adenosine transport, also reduced Na+ transport. Taken together, the data suggest that 2-CA inhibits Na+ transport by interfering with adenosine transport or metabolism.     

Intracellular Na+ regulates dopamine and angiotensin II receptors availability at the plasma membrane and their cellular responses in renal epithelia

The balance and cross-talk between natruretic and antinatruretic hormone receptors plays a critical role in the regulation of renal Na+ homeostasis, which is a major determinant of blood pressure. Dopamine and angiotensin II have antagonistic effects on renal Na+ and water excretion, which involves regulation of the Na+,K+-ATPase activity. Herein we demonstrate that angiotensin II (Ang II) stimulation of AT1 receptors in proximal tubule cells induces the recruitment of Na+,K+-ATPase molecules to the plasmalemma, in a process mediated by protein kinase Cbeta and interaction of the Na+,K+-ATPase with adaptor protein 1. Ang II stimulation led to phosphorylation of the alpha subunit Ser-11 and Ser-18 residues, and substitution of these amino acids with alanine residues completely abolished the Ang II-induced stimulation of Na+,K+-ATPase-mediated Rb+ transport. Thus, for Ang II-dependent stimulation of Na+,K+-ATPase activity, phosphorylation of these serine residues is essential and may constitute a triggering signal for recruitment of Na+,K+-ATPase molecules to the plasma membrane. When cells were treated simultaneously with saturating concentrations of dopamine and Ang II, either activation or inhibition of the Na+,K+-ATPase activity was produced dependent on the intracellular Na+ concentration, which was varied in a very narrow physiological range (9-19 mm). A small increase in intracellular Na+ concentrations induces the recruitment of D1 receptors to the plasma membrane and a reduction in plasma membrane AT1 receptors. Thus, one or more proteins may act as an intracellular Na+ concentration sensor and play a major regulatory role on the effect of hormones that regulate proximal tubule Na+ reabsorption.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures

HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells. Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells. For polio antibody titration, HEp-2 and Vero cells were equally suitable. A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C. Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests.     

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction.