Induction of an interferon-like substance in persistently infected Aedes albopictus cells

Summary The multiplication of Sindbis virus inSingh'S mosquito cell line derived from larvalA. albopictus was studied. Persistently infected cells are not able to support the growth of Sindbis virus to the same extent as cells infected for the first time. The maintenance of cell-virus equilibrium in persistently infected cells seems to be due to the presence of interferon-like antiviral substances. Mosquito cells which had been treated with media harvested from persistently infected cultures were protected from infection by Sindbis virus. The synthesis of these interferon-like substances is inhibited by actinomycin D. A possible implication of this observation is that in persistently infected mosquito cells the rate of replication of virus is controlled by the genome of the host cell.     

Inhibitors of IMP dehydrogenase prevent sindbis virus replication and reduce GTP levels in Aedes albopictus cells

We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction (80–90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 μg/ml), and (3) α-amanitin (10 μg/ml) in α-amanitin-sensitive cells but not in α-amanitin-resistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed.     

Sindbis virus infection increases hexose transport in quiescent cells

Sindbis virus infection of baby hamster kidney cells or chick embryo cells resulted in a significant increase in the rate of uptake of [2-3H]deoxy-D-glucose ([3H]dGlu). Stimulation of hexose transport in Sindbis virus-infected cells occurred only if the cells were rendered quiescent by culturing at high density or by serum starvation. In contrast, Sindbis virus-induced inhibition of potassium transport, measured as a decrease in the uptake of 86Rb+, was independent of cell growth state. Stimulation of [3H]dGlu uptake in Sindbis virus-infected cells was the result of an increase in the Vmax of the hexose transporter, but not a change in the Km. The stimulation of [3H]dGlu uptake induced by Sindbis virus was insensitive to the drug actinomycin D, but was blocked by cordycepin. The stimulation was also insensitive to treatment with tunicamycin, which prevented the virally induced inhibition of the plasma membrane-associated Na+/K+ ATPase and termination of host protein synthesis.     

Growth and release of several alphaviruses in chick and BHK cells

The growth and release of several alphaviruses, including several strains of Sindbis virus (the wild-type strain, the large plaque and small plaque variants of the HR strain, and the HR mutant ts103), Semliki Forest virus(SFV) and Middelburg virus, and of the unrelated rhabdovirus, vesicular stomatitis virus (VSV), have been compared in chick cells and in BHK-21 cells as a function of the culture conditions for the host cell and the ionic strength of the medium. The small plaque strain of Sindbis HR, as well as SFV, grew better in BHK cells, whereas the large plaque strain of Sindbis HR showed a preference for chick cells. Wild-type Sindbis and VSV grew equally well in either cell. The optimum ionic strength for virus production as well as inhibition of virus release into the medium at low ionic strength depended upon both the virus and the host cell. Thus, VSV grown in medium of low ionic strength gave no additional release of virus on incubation with hypertonic medium (minimum effect), whereas ts103 released very little virus without exposure to hypertonic conditions (maximum effect). The viruses could be ordered as follows: minimum effect = vesicular stomatitis virus < Middelburg virus < Semliki Forest virus < Sindbis wt < Sindbis HR (large plaque) < Sindbis HR (small plaque) < Sindbis ts103 = maximum effect. After several passages in culture, chick cells required hypertonic conditions for optimum production and release of Sindbis virus. Furthermore, BHK cells cultured in different media responded differently to ionic strength for virus production and release. These results suggest that there is a charge-dependent stop in the maturation of alpha-viruses, possibly a configurational rearrangement of glycoprotein E2 upon its formation from the precursor PE2, which is sensitive to the ionic strength of the medium, to the composition of the host plasmalemma and to differences in the virus glycoproteins.     

Comparative studies of the 3''-terminal sequences of several alpha virus RNAs

We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction (80–90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 μg/ml), and (3) α-amanitin (10 μg/ml) in α-amanitin-sensitive cells but not in α-amanitin-resistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed.     

Virazole prevents production of sindbis virus and virus-induced cytopathic effect in Aedes albopictus cells

The nucleoside analogue virazole (1-β-d-ribofuranosyl 1,2,4-triazole-3-carboxamide) at a concentration of 150 μg/ml inhibited the production of Sindbis virus (SV) and prevented virus-induced cytopathic effect (CPE) in Aedes albopictus LT C-7 cells. The drug had no effect on net viral RNA synthesis, the production of infectious RNA, or on nucleocapsid assembly. All viral structural proteins (E₁, E₂, and C) were synthesized and glycosylated in apparently normal amounts in the presence of virazole (Vz). However, most cells contained little or no reactive viral antigens as measured by an indirect immunofluorescent test. In contrast to the minimal effect on the synthesis of viral RNA and proteins, Vz markedly inhibited host macromolecular synthesis, as well as the growth of mosquito cells. In BHK21 cells the situation was different. As in C-7 cells, the drug caused a marked reduction in host macromolecular synthesis. However, CPE was not prevented and virus production was not inhibited. It is suggested that certain host functions may be required in the late stages of viral maturation and for expression of CPE and that these host functions are inhibited by Vz.     

Replication of echo virus type 25 JV-4 reference strain and wild type strains in MRC5 cells compared with that of poliovirus type 1

Summary In echo virus type 25/JV-4 the shut off of host cell protein synthesis took significantly longer and the kinetics of the synthesis of viral proteins and viral RNA occurred much later than in the poliovirus. However, these characteristics impaired neither polyprotein processing nor virus production in the JV-4 strain. In contrast the two wild strains M.1262 and Th.222 had a lower virus yield than strain JV-4. The presence of a high Mr protein in the pattern of viral proteins of wild strains suggested that a defect in the polyprotein processing was responsible for the decreased virus yield. The infectious cycle of strain Th.222 differed from that of strains JV-4 and M.1262 in the rapid inhibition of host cell translation and the extent of viral protein synthesis. The sensitivity to actinomycin D was also investigated. Strain M.1262 was found to be insensitive. The virus yield of strains JV-4 and Th.222 was three- and fourfold lower respectively in the presence of actinomycin D. This sensitivity to the antibiotic was observed during viral RNA synthesis in strain JV-4 and during viral protein synthesis in strain Th.222. These results suggest that cellular factors are involved in the replication of echo virus type 25 strains in MRC5 cells.     

The effect of actinomycin D on the synthesis of influenza virus-specific RNA

Summary The effect of actinomycin D on the replication of CAM strain of influenza A1 virus was studied in the human conjunctival cell line, clone 1-5C-4. Actinomycin D in a concentration of 0.5 mcg/ml suppressed the virus yield to less than l/1000th of that from the uninhibited culture. When the addition of actinomycin D was delayed, the resistance of the production of virus and viral components to actinomycin D developed progressively in the order of the soluble antigen, hemagglutinin, and the infective virus. The synthesis of virus-specific RNA was demonstrable by the incorporation of tritiated uridine when actinomycin D was added to the culture at 6 hours after infection, but not when it was added at 1 hour after infection. These findings indicated that actinomycin D illhibits influenza virus replication because it inhibits the synthesis of virusspecific RNA and that, however, it does not directly block virus-specific RNA synthesisper se but exerts the inhibitory effect on some event(s) preceding virus-specific RNA synthesis. In FL cells, in which CAM virus undergoes an abortive multiplication cycle, virus-specific RNA was synthesized in the same period after infection and in a similar quantity as in clone 1-5C-4 cells in which complete multiplication occurs.     

Multiplication du virus Sindbis dans des cellules de Drosophile cultivées in vitro

Résumé Les données présentées montrent qu'il s'établit entre le virus Sindbis et les cellules de Drosophile en culture un système d'infection persistante comme il s'en établit un entre les autres cellules d'insecte et tous les Arbovirus.L'évolution de la production cellulaire moyenne a pu être précisée dans une expérience: on constate un maximum de la production environ 24 heures après l'infection, puis la production se ralentit et se fixe à un niveau d'équilibre qui se maintient indéfiniment à travers les divisions cellulaires.

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Multiplication of Sindbis virus in Drosophila cells cultivated in vitro

Summary Sindbis virus replicates in Drosophila cell cultures without any cytopathic effect. In continuous cell lines the virus is able to establish a persistent infection similar to other arboviruses in insect cell lines.The growth curve of Sindbis virus in Drosophila cells shows a maximum virus yield at around 24 hours postinfection, thereafter the virus production decreases and then remains fairly constant during a number of cell divisions. The average virus yield per cell has been estimated to be small,i.e. at best 5 PFU per day.

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Avec 4 Figures

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Cet article fait partie d'un mémoire qui sera présenté en vue de l'obtention du doctorat ès Sciences devant l'Université de Paris XI en 1975.     

Enhanced interferon production from chick embryo cells aged in in vitro

The plaque-forming efficiency of Sindbis virus decreased as much as 1000-fold, and plaque size was diminished markedly, when tested on chick embryo cell monolayers aged in vitro. The plaquing efficiency and plaque size of Newcastle disease virus was unaffected. The reaction(s) associated with aging in vitro which lead to lowered plaquing efficiency are slowed considerably when cell monolayers are: (1) established in Simpson-Hirst medium rather than standard growth medium; (2) held at 31 degrees ; (3) given a regimen of daily medium changes; or (4) trypsinized and used as young secondary cultures. A loss in the average yield of virus per cell accompanies the loss in plaquing efficiency of Sindbis virus on aged monolayers. Adding actinomycin D to the aged cells at the time of infection eliminated completely the inhibition of Sindbis virus replication. Cells aged for 7 days in vitro were found to produce up to 32 times more interferon than cells 1-2 days old and were more sensitive to the action of interferon. The decrease in efficiency of Sindbis virus plaquing and yield in aged cells is accounted for by their development of an enhanced capacity to synthesize interferon upon appropriate stimulation. The process of contact inhibition and its concomitant regulation of macromolecular synthesis seems implicated in the aging phenomenon in that it may produce a generalized state of "enhanced derepressibility" in the cell.     

The inhibition of cowpea mosaic virus replication by actinomycin D.

The effect of actinomycin D on the multiplication of cowpea mosaic virus (CPMV) in cowpea mesophyll protoplasts was studied. Using 10 μg/ml, a concentration at which actinomycin D reduced total RNA synthesis by more than 90%, propagation of CPMV was inhibited completely, provided the drug was present from the time of inoculation. The degree of inhibition of CPMV multiplication by actinomycin D progressively decreased with time after inoculation. At 8 hr after inoculation, a stage at which the infection is still in its latent phase, multiplication had become resistant to the antibiotic. Even under conditions of complete inhibition of virus replication, synthesis of virus antigen still continued as measured by staining of infected protoplasts with fluorescent antibodies made against purified virus. It was demonstrated that under these circumstances de novo production of viral top component (empty capsids) continued. Furthermore six virus-specific polypeptides were detected in CPMV-infected protoplasts. Their molecular weights were estimated to be 170,000; 110,000; 84,000; 37,000; 22,000; and 21,000. Of these the 37,000- and 22,000-molecular weight proteins probably represent the viral capsid proteins. Since all proteins were synthesized upon inhibition of host RNA synthesis by actinomycin D, it is concluded that they are specified by the viral genome.     

Effect of ouabain on Sindbis virus replication in ouabain-sensitive and ouabain-resistant Aedes albopictus cells (Singh)

The effects of ouabain on Sindbis virus replication in cloned sublines of ouabain-sensitive (B₁C₄) and -resistant (Oua₅) Aedes albopictus cells have been studied. The addition of ouabain (0.1 mM) to B₁C₄ cells 1 hr after infection reduced the yield of infectious virus 500-fold. The same concentration had no effect on the yield from Oua.5 cells. Analysis of [³H]-uridine-labeled RNA by gel electrophoresis showed that ouabain markedly inhibited Sindbis virus-specific RNA synthesis in B₁C₄ cells but only partially inhibited host RNA synthesis. In Oua₅ cells, ouabain had little effect on either host or viral RNA synthesis. A close correlation was observed between the inhibition of host protein synthesis by ouabain and the inhibition of virus replication.     

Effects of collagen matrix on Sindbis virus infection of BHK cells

The effects of extracellular matrix (ECM) components on the outcomes of alphavirus interaction with cells are not known. Studies that address such interactions have to address several methodological difficulties, including: the survival of the cells within the matrix; the passage of the virus through the matrix to infect embedded cells; and the dissociation of cells and matrix into single-cell suspension, before and after virus infection, for quantitative analysis. Herein, these issues were addressed in the context of a model system of collagen as the ECM component, baby hamster kidney (BHK) cells, and Sindbis virus. The outcomes of Sindbis virus infection of BHK cells, grown in three-dimensional (3D) collagen gel versus on plastic, and on two-dimensional (2D) collagen versus bovine serum albumin (BSA)-coated surfaces were compared. Cell morphology was more slender in 3D and on 2D collagen than on plastic or BSA-coated surfaces. The cells were able to survive in the 3D environment. Using Sindbis virus carrying the green fluorescent protein gene, the virions were found to be capable of penetrating the 3D collagen matrix and infecting the cells. There was more infectious virus in cultures of cells in 3D and on 2D collagen than on plastic or BSA-coated surfaces, respectively. Higher virus titers from cells on 2D collagen compared to BSA-coated surfaces was not associated with uninfected cell number or viability but with increased cell survival after infection. Infected cells on BSA surfaces became detached, while those on 2D collagen remained attached. These experiments establish procedures for analysis of interaction of collagen, BHK cells, and Sindbis virus and suggest that collagen increases infectious Sindbis virus titers from BHK cells by enhancing post-infection cell survival.     

Substitution in the poliovirus replicase gene determines actinomycin D sensitivity of viral replication at elevated temperature

A series of ts+ revertants and recombinants derived from a temperature-sensitive plurimutant of poliovirus type 1 showed identical plaquing efficiencies at 37 degrees C and at 39 degrees C and exhibited similar yields and plaque morphology to wild-type virus. However, these viruses were characterized by clear inhibition of viral RNA synthesis at 39 degrees C, as measured by uridine incorporation in the presence of actinomycin D. Similarly, virus yields were decreased by one log in the presence of actinomycin D during infection at 39 degrees C. All the ts+ recombinants formed between temperature-sensitive mutants of poliovirus that were inhibited by actinomycin D carried a glutamine----histidine modification at residue 170 of their viral replicase (polypeptide 3D), due to a G----U substitution at nucleotide 6496. Inhibition of viral growth was increased by pretreatment of cells with actinomycin D for 3 h prior to infection, suggesting that actinomycin D sensitivity could reflect an increased dependence of viral RNA replication on host factor(s).     

In vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3

Summary Thein vitro virus yield of MHV3 reached 10⁷ PFU/ml in mouse DBT cells infected with a virus suspension in HEPES-buffered medium containing DEAE-dextran. The virus titer was 10⁶ PFU/ml in the presence of 10 µg actinomycin D/ml. MHV3 grown in DBT cells gave three peaks of density (1.10–1.14 g/cm³, 1.18–1.20 g/cm³, and 1.25–1.31 g/cm³) in sucrose gradients. All these peaks retained infectivity.With 2 Figures     

Influence of actinomycin D on the effectiveness of producing ts mutants of the Japanese encephalitis virus

Reciprocal competition of viral and cellular matrices of RNA synthesis was demonstrated, as well as a 10-13-fold increase of 3H-uridine incorporation into viral RNAs in the presence of actinomycin D, the inhibiting effect of actinomycin D on virus replication, the dependence of the late stage of Japanese encephalitis virus reproduction upon the host cell, and no positive effect of actinomycin D on generation of Japanese encephalitis virus ts mutants.     

Interferon production by individual cells

A feline interferon production and assay system has been defined which allows measurement of the interferon produced by a single cell. It was found that 2–3 induced cells were required to reduce by 0.5 log¹⁰ the yield of vesicular stomatitis virus (VSV) from approximately 2500 target cells. The antiviral effect was characterized as interferon by several criteria. The presence of trypsin or pretreatment of the induced cells with actinomycin D eliminated the effect. The antiviral effect was species specific, and cell-free supernatants from target cell monolayers which had been exposed to induced cells could be used to protect other target cell monolayers. It was found that one interferon-producing cell provided significant (p < 0.05) 0.1 log₁₀ reduction of VSV yield from 2500 target cells. Analysis of the data obtained when 2 or 3 induced cells were used to provide antiviral protection indicated that approximately $\text{1}\text{3}$ of the cell population failed to respond measurably to the inducer. The number of cells required to protect target cells was found to be inversely proportional to the amount of interferon produced in monolayer culture.     

Protein synthesis in Japanese encephalitis virus-infected cells

We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK₂ (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK₂ cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971). In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones.We then studied the effects of puromycin and cycloheximide in LLC-MK₂ cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK₂ cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.