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.     

Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis

Bolognesi, D. P. (Rensselaer Polytechnic Institute, Troy, N.Y.), and D. E. Wilson. Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis. J. Bacteriol. 91:1896-1901. 1966.-Infection by Newcastle disease virus brings about a rapid and marked inhibition of cell protein synthesis (CPS) in chick embryo fibroblast monolayers. The block to CPS is initiated about 5 hr after infection, and by 9 hr about 85% of the host protein synthesis is shut off. Azauridine (3 mg/ml), a ribonucleic acid (RNA) synthesis inhibitor, prevents the virus-induced inhibition of CPS when added at the time of infection; but it does not prevent the inhibition when added at 3 hr after infection. When puromycin (60 mug/ml), a protein synthesis inhibitor, was added at 3.5 hr after infection, viral RNA was synthesized in normal amounts, but the virus-induced inhibition of CPS was prevented. Actinomycin D added at the time of infection does not, however, prevent the virus-induced inhibition of CPS. The results of these experiments indicate that proteins synthesized during Newcastle disease virus replication are responsible for the inhibition of host-cell protein synthesis. The synthesis of these inhibitory proteins depends on the prior synthesis of viral RNA.     

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.     

Effect of Mengovirus Replication on Choline Metabolism and Membrane Formation in Novikoff Hepatoma Cells

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.     

Inhibition of Mitosis and Macromolecular Synthesis in Rat Embryo Cells by Kilham Rat Virus

The effects of Kilham rat virus multiplication were studied in cultured rat embryo cells to examine the mechanisms by which virus infection might be related to developmental defects in rats and hamsters. The virus was found to inhibit motosis and deoxyribonucleic acid (DNA) synthesis within 2 to 10 hr after infection. However, total ribonucleic acid synthesis was relatively unaffected until about 20 hr after infection, and total protein synthesis did not decline significantly until loss of viable cells was apparent in the cultures. No effect on chromosomes was detected. The effect of Kilham rat virus on DNA synthesis appears to be due to inhibition of macromolecular synthesis rather than to an inhibition of uptake of precursors into cells. The effect of the virus on mitosis may be an addition to the effect on DNA synthesis, since mitosis is inhibited even in cultures in which cells are able to divide at the time of infection and which have presumably completed DNA synthesis.     

Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells.

In this article, we describe two distinct effects of cycloheximide (CH), a potent inhibitior of protein synthesis, on the replication of polyoma virus (PV) DNA in an inducible line of PV-transformed rat cells (LPT cells). Exposure of LPT cells to CH causes up to an 8 fold increase in the cellular concentration of PV DNA determined by molecular hybridization. The same treatment inhibits cell division and chromosomal DNA replication. However, the amount of chromosomal DNA per cell is not affected by the drug. In LPT cells treated with mitomycin C (MMC), PV DNA replication is enhanced after 7 hr. During the period extending from 7 hr to 24 hr, the concentration of virus DNA increases at least 100 fold. CH added to the cells 0-7 hr after treatment with MMC inhibits the replication of PV DNA by 90-100%. The inhibition is less effective in cells exposed to CH from 7 hr and on. The inhibitory effect is reversible: virus DNA synthesis is resumed after removal of CH from the growth medium. Thus CH acts as an inducer of virus DNA synthesis in cells whose resident viral genome is repressed, but inhibits the autonomous replication of the activated genome following induction with MMC.     

Growth Characteristics of Kilham Rat Virus and Its Effect on Cellular Macromolecular Synthesis

Kilham rat virus (KRV) is adsorbed into the rat nephroma cell within 1 hr after infection. There follows a latent period of about 12 hr during which less than 1% of the input infectious virus can be accounted for. New infectious virions can be detected at about 12 hr and the maximal yield of virus is attained by 23 hr after infection. The increase in final virus yield is about 200-fold over that found in the latent period. During this 23-hr period of virus growth, the rate of protein synthesis remains 75 to 100% of that in the uninfected cell. Ribonucleic acid (RNA) synthesis during this period is maintained at 100 to 150% of that found in the control cells. The addition of the inhibitor of deoxyribonucleic acid (DNA) synthesis, 5-fluoro-deoxyuridine (FUDR), up to 8 hr after infection completely suppresses virus production. After 8 hr, viral DNA production has started and FUDR inhibition progressively decreases until by 23 hr the addition of the inhibitor no longer causes a reduced virus yield. Viral DNA synthesis once initiated is required for the remainder of the 23-hr virus cycle. Viral DNA synthesis probably begins about 4 hr before the production of infectious virions. In the KRV-infected cells, DNA synthesis decreased sharply for 6 to 7 hr after infection in comparison to the uninfected cell. At 7 to 8 hr after infection, DNA synthesis in the infected cell increased and was maintained at a higher level than in the control cells for the rest of the virus growth period.     

Synthesis of Ribonucleic Acid in Cells Infected with LSc Poliovirus at Elevated Temperatures

The synthesis of viral ribonucleic acid (RNA) was detected within 2 hr after infection with LSc poliovirus at 35 C. This RNA eluted as a single peak with 0.9 m NaCl on methylated albumin celite columns, was sensitive to ribonuclease, precipitated in the presence of 2 m LiCl, and had an S(20) value at 34 +/- 2 in linear sucrose gradients. When cells were infected at 39 to 40 C, there was also early synthesis of RNA. However, 2 hr after infection this synthesis was drastically inhibited. The absence of net RNA synthesis at 39 to 40 C during the late stages of infection was not caused by rapid degradation of newly formed RNA, since the RNA produced between 1 and 2 hr at 39 to 40 C was still present 3.5 hr after infection. There was a 3 log(10) inhibition in the production of infectious virus when p-fluorophenylalanine was present in the medium at a concentration of 25 mug/ml. This concentration of analogue had little effect upon the production of viral polymerase and viral RNA. Virus grown in the presence of analogue at a concentration of 10 mug/ml exhibited increased heat sensitivity compared to control virus. However, viral polymerase exhibited no change in sensitivity to heat or manganese when cells were exposed to 25 mug of p-fluorophenylalanine per ml during infection. p-Fluorophenylalanine had a relatively selective effect on viral capsid protein but did not reverse the inhibition of synthesis of viral RNA at 39 to 40 C.     

Effect of Polyions on the Infectivity of Rabies Virus in Tissue Culture: Construction of a Single-Cycle Growth Curve

The infectivity of fixed rabies virus in a number of cell lines has been shown to be markedly enhanced by the addition of protamine or diethylaminoethyl dextran to the virus inoculum. The polycations appear to exert their influence at a very early stage (adsorption or penetration or both) of virus-cell interaction. Immune globulin blocked infection completely when added up to 5 min after exposure and almost completely when added 5 to 15 min after infection. Antibody had no effect on adsorption and penetration when added to the inoculum 30 min or more after cells were exposed to the virus. Irradiation of BHK/21 cell monolayers with ultraviolet light increased their sensitivity to rabies virus. The events occurring after synchronous infection of cells in both irradiated and nonirradiated cell monolayers were followed by means of fluorescent-antibody staining and by intracerebral titration in mice. Virus-specific fluorescent antigen first appeared between 8 and 9 hr after infection, and in irradiated cultures there was a further lag period of 3 hr before infectious virus was produced intracellularly. Virus was first detected in the medium 12 to 15 hr after infection, and maximal yield of infectious virus was observed 48 hr after exposure. In nonirradiated cultures, formation of infectious virus was delayed, and the final yield of virus was also reduced.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Transformation by Murine Sarcoma Virus: Fixation (Deoxyribonucleic Acid Synthesis) and Development

Transformation of rat embryo cells by murine sarcoma virus (MSV) was contingent upon synthesis of deoxyribonucleic acid (DNA) during the first 12 hr of infection. Inhibition of DNA synthesis by thymidine (20 mm) or cytosine arabinoside (0.1 mm) resulted in the protection of cells from transformation by MSV. Transient suppression of DNA synthesis prior to infection or after a 12-hr delay had little effect on subsequent transformation, emphasizing the critical time period in in which DNA synthesis was necessary for intracellular fixation of the viral genome. These results are similar to those previously described for Rous sarcoma virus. Development of transformed cells after viral fixation was shown to be influenced by cellular density. Under conditions which allowed fixation of virus in confluent cellular monolayers, less than 20% of these cells developed into transformed foci.     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Irreversible Effects of Cycloheximide During the Early Period of Vaccinia Virus Replication

The presence of cycloheximide, an inhibitor of protein synthesis, during the period 30 to 60 min after vaccinia infection produced an irreversible block in virus replication. In contrast (i) cycloheximide given at earlier or later times, even for prolonged periods, did not prevent continuation of the infectious cycle after removal of the drug, and (ii) treatment with cycloheximide during the first 2 hr did not prevent virus growth when the early stages of replication proceeded more slowly due to infection with a low multiplicity of virus. These findings were interpreted as an indication that protein synthesis is required at a critical time in the virus growth cycle. Under the conditions in which brief cycloheximide treatment prevented virus growth, ribonucleic acid (RNA) synthesis continued at an undiminished rate for at least 2 hr after removal of the drug. Although this RNA appeared identical by polyacrylamide gel electrophoresis to "early" viral messenger RNA, it was not found associated with ribosomes or polyribosomes. Failure to observe viral protein synthesis was consistent with the latter finding. It appeared unlikely that the translational block resulted from inadequate removal of cycloheximide, since the effects of the drug were shown to be reversible at earlier or later times in infection or even at the same time when a lower multiplicity of virus was used. Interference with the normal synthesis of specific viral protein factors required for translation was postulated to explain the results.     

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

Analysis of Simian Virus 40-Induced Transformation of Hamster Kidney Tissue in Vitro VIII. Induction of Infectious Simian Virus 40 from Virogenic Transformed Hamster Cells by Amino Acid Deprivation or Cycloheximide Treatment

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.     

Antiviral Effect of Cyclic Amines on a 1969 Isolate of A2 Influenza

Cyclooctylamine and amantadine inhibit the growth of 1969 isolates of A(2) influenza virus to a significant degree. There was slightly more inhibition of the virus by the cyclooctylamine (COA) than the amantadine; however, the dose of COA used was greater than the dose of amantadine. There was no significant difference between flasks treated 3 or 4 hr and those treated 2 hr. However, there was a curious relationship of more plaques in the flasks exposed to the two drugs for the longer intervals. Other experiments done with slight modifications in technique support the antiviral effect demonstrated in this experiment when the cell system is pretreated prior to virus infection. In two experiments, pretreating the cells for 2 hr with COA at 100 mug/ml but removing the drug solution and washing the cells prior to virus inoculation revealed no differences in plaque counts between controls and treated cells. This would indicate that the antiviral effect required the presence of the drug during the early stages of penetration of the cells by the virus particles.     

Replication of canine herpesvirus: I. Synthesis of viral deoxyribonucleic Acid

This paper reports the results of two series of experiments. The first series indicated that deoxyribonucleic acid (DNA) extracted from partially purified canine herpesvirus virions is characterized by a high guanine plus cytosine molar base ratio (65 to 67 mole%), similar to the DNA of herpes simplex virus. In the second series of experiments it was estimated, on the basis of uptake of tritiated thymidine, that in dog kidney cells canine herpesvirus-DNA synthesis starts at 4 hr and continues until 16 hr after infection. Treatment of infected cells with puromycin during the first 4 hr of infection blocks the onset of viral DNA synthesis, whereas, after this time the uptake of thymidine is unaffected.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Kinetics of Viral Deoxyribonucleic Acid, Protein, and Infectious Particle Production and Alterations in Host Macromolecular Syntheses in Equine Abortion (Herpes) Virus-infected Cells

Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of viral DNA and protein synthesis and assembly into mature virus was determined with the inhibitors 5-fluorodeoxyuridine (FUdR) and cycloheximide, respectively. Thus, viral DNA synthesis was essentially completed at 14 hr, and viral protein and infectious virus synthesis was completed at 18 hr. Although the number of plaque-forming units (PFU) produced by FUdR-treated cells (10(3) to 10(4) PFU/ml) was at least 3 logs less than that produced by untreated cells, the yield of physical particles (as determined by electron microscopy) was approximately the same at 30 hr after infection. Besides being relatively non-infective, the particles produced in FUdR-treated cells appeared morphologically incomplete as they contained little or no nucleoid material.