Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.     

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone.

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.     

Effect of colchicine on virus-induced interferon synthesis in chick embryo cells

Colchicine, at a concentration of 5 X 10(-4) M (0.2 microgram/ml), inhibits interferon synthesis induced by theLee strain of influenza B virus in chick embryo cells, but it does not influence the release of preformed interferon from cells. The same drug concentration does not affect the overall synthesis of cellular RNA and protein. The inhibition of interferon synthesis by colchicine is a temperature-dependent process and is not manifested at 0 degrees C. Colchicine is found to be most effective when it is introduced into the medium at early stages of infection. It is suggested that colchicine inhibits the formation of messenger RNA for interferonogenesis.     

Evidence for Clostridium perfringens Enterotoxin (CPE) Inducing a Mitogenic and Cytokine Response In Vitro and a Cytokine Response In Vivo

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MΦ) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MΦ were concurrently incubated with CPE and interferon gamma (IFN-γ). MΦ incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-γ, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4–8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms. Received: 25 April 1998 / Accepted: 2 September 1998     

Mechanism of Mengo Virus-Induced Cell Injury in L Cells: Use of Inhibitors of Protein Synthesis to Dissociate Virus-Specific Events

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.     

Role of Interferon in the Propagation of MM Virus in L Cells

MM virus propagated in mouse brain replicates to low titers in L cells without production of cytopathic effect (CPE). After growing the virus in BHK-21 cells, however, the virus replicates to high titers in L cells with complete CPE. It was found that suspensions of MM virus propagated in L cells directly from the mouse brain contained much more interferon than did suspensions of virus which had first been grown in BHK-21 cells. Mouse brain suspensions of the virus were also found to contain high interferon titers. Treatment of L cells with actinomycin D before infection with mouse brain-grown virus resulted in full virus replication with CPE. BHK-21 cell-grown virus diluted in L cell interferon behaved like mouse brain-grown virus in L cells. It is concluded that the presence of interferon in the inoculum is largely responsible for the suppression of MM virus replication in L cells.     

Interferon-associated, dsRNA-dependent enzyme activities in a mutant 3T6 cell engaged in the semiconstitutive synthesis of interferon

Cytoplasmic extracts of untreated cultures of a virus-resistant mutant of mouse 3T6 cells, designated 3T6-VrB2, contain two double-stranded, RNA-activated enzyme activities associated with interferon action. These are the synthesis of a low molecular weight oligonucleotide inhibitor of cell-free protein synthesis from ATP, and the phosphorylation of a 67,000 dalton polypeptide by transfer of the gamma phosphate of ATP. Basal levels of both enzyme activities are detectable in extracts of untreated parental 3T6 cells, and are greatly enhanced upon interferon pretreatment. A procedure was developed, using a nonionic detergent to effect cell lysis, which allowed the analysis of the protein kinase activity from as few as 2 x 10(7) cells. Using this procedure, direct proportionalities were demonstrated between the concentration of interferon to which 3T6 cells were exposed, and both the level of protein kinase activity and the magnitude of the antiviral state were established in these cells. Furthermore, untreated cultures of 3T6-VrB2 exhibited both an antiviral state and an intracellular protein kinase activity equal to that of cultures of the parental 3T6 cells pretreated with a single concentration of mouse interferon.     

Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.     

New simple dye-uptake assay for interferon

Using the spectrophotometer that the authors developed, the amounts of human leukocyte and mouse L cell interferons on FL cells and L929 cells were measured and values were compared with those measured by the cytopathogenic effect (CPE) reduction method (CPE method). The spectrophotometric method, which was simpler than the original dye-uptake method, was found to be more sensitive than the latter. When Sindbis virus was used instead of vesicular stomatitis virus (VSV), there were no significant differences in the sensitivities of the two methods or the interferon titers estimated. When FL cells or L929 cells were treated with interferon at the time of their dispersion, their interferon titers were almost the same as those of cells treated with interferon 2 days after dispersion. It is concluded that this new dye-uptake method is useful for assay of human and mouse interferons.     

Effect of interferonogenic molecular complex of yeast RNA--tilorone on DNA, RNA and protein synthesis in vitro

In the experiments in vitro using the primary mononuclear cells (MNC) culture of the human peripheral blood the influence of interferonogenic yeast RNA-tilorone molecular complex on the DNA, RNA and protein synthesis was studied. The complex was shown to inhibit the insertion of 3H-thymidine, 3H-uridine and 3H-leucine into DNA, RNA and protein of MNC total pool (by 13, 1 and 40% respectively); that was practically conformed with this synthesis inhibition upon to a natural origin polynucleotide interferon inducers--lariphan (9, 0 and 57% respectively) and ridostin (9, 0 and 56% respectively) action, and at the same time rather less than poly(I)-poly(C) (14, 5 and 62% respectively). In the case of preliminary cell stimulation by the mitogen PHA the complex revealed comitogenic action at a concentration 25 micrograms/ml, that corresponded to optimal for interferonogenesis; the increase of the doses till 100-1000 micrograms/ml lead to in the reversal effect. To proceed from mutual relation between interferonogen preparations influence on the mentioned synthesis and their cytotoxicity the conclusion was about made the complex promising usage as an interferon inducer both in vitro and in vivo conditions.     

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.     

Enterotoxic strains of Clostridium perfringens and sporulation

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.     

Effect of Sindbis virus infection on induction of heat shock proteins in Aedes albopictus cells.

When Aedes albopictus cells (clone C7) were infected with Sindbis virus, the production of cytopathic effect CPE depended largely on the conditions under which the cells were cultured. We observed marked inhibition of cellular RNA and protein synthesis, as well as a loss of the ability to induce heat shock proteins, e.g., hsp70, under conditions which led to cytopathic effect. Infected cells in which heat shock proteins could no longer be induced contained much lower amounts of hsp70 mRNA after heat shock than did mock-infected cells which were similarly treated. It is suggested that this decreased level of hsp70 mRNA is due to a failure of these cells to synthesize hsp70 mRNA after heat shock.     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

The effects of Clostridium perfringens enterotoxin on intracellular levels or transport of uridine, thymidine and leucine do not fully explain enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

Clostridium perfringens type A enterotoxin (CPE) has been shown previously to inhibit the incorporation of radiolabeled precursors into acid-insoluble material but the mechanism of inhibition is unknown. It has also been shown that extracellular calcium is required for some CPE effects. In this report, it is shown that CPE completely and virtually simultaneously inhibits incorporation of precursors into RNA, DNA and protein in either the presence or absence of extracellular divalent cations and that changes in intracellular precursor levels did not consistently correlate with this CPE-induced inhibition of incorporation. These results strongly suggest that CPE can inhibit macromolecular synthesis, not just inhibit precursor transport. It is inferred from this that CPE can affect DNA and RNA synthesis, and possibly protein synthesis, by altering other cellular processes besides, or in addition to, precursor transport and these effects then lead to a shutdown of macromolecular synthesis.     

Interferon production in chick-embryo cells. The effect of puromycin and p-fluorophenylalanine

1. When chick-embryo cells were treated with ultraviolet-inactivated influenza virus (Melbourne strain), interferon was produced after a lag period of about 10hr. 2. The addition of small amounts of either puromycin or p-fluorophenylalanine immediately after the virus inhibited the subsequent production of interferon. Both inhibitors primarily affected protein synthesis, and it is concluded that interferon production involves new protein synthesis. 3. Results obtained by the addition of either inhibitor for short periods during the lag phase demonstrated a requirement for protein synthesis during the second half of the lag phase. 4. Addition of puromycin during the course of interferon production caused almost immediate inhibition, but interferon formation became insusceptible to the action of p-fluorophenylalanine at about 26hr. after infection. Possible explanations of this effect are discussed.     

Differential Effects of a Phorbol Ester on Carboxypeptidase E in Cultured Astrocytes and AtT-20 Cells, a Neuroendocrine Cell Line

Abstract: Cultured astrocytes have been shown to secrete various neuropeptides and the neuropeptide processing enzyme, carboxypeptidase E (CPE). The secretion of CPE enzymatic activity from astrocytes has been shown previously to be increased approximately twofold by treatment with tetradecanoylphorbol 13-acetate (TPA), a phorbol ester. In this study, metabolic labeling with [³⁵S]Met was utilized to examine the effect of TPA on the biosynthesis of CPE protein in cultured astrocytes and in AtT-20 cells, a pituitary-derived cell line. Treatment of astrocytes with 0.1 μg/ml TPA for 24 h caused an 80% increase in the level of radiolabeled CPE in both the media and the cells, indicating that the synthesis of CPE was stimulated by the TPA. AtT-20 cells also secreted more radiolabeled CPE in response to TPA, but this increase was offset by a proportional decrease in the cellular level of radiolabeled CPE, and synthesis of CPE was not stimulated in this cell line. Northern blot analysis demonstrated that 0.1 μg/ml TPA elevated CPE mRNA by approximately 50% in cultured astrocytes but not in AtT-20 cells. Quantitative in situ hybridization studies demonstrated that the TPA-induced increase in CPE mRNA expression was largely due to increases in the number of cells expressing CPE mRNA, although for astrocytes from some brain regions the average level of CPE mRNA per cell was also elevated by TPA. These results suggest that astrocytes can be induced to express CPE, which is consistent with a role for astrocytes in intercellular signaling.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.