Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells

Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1/S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.     

Inhibition of the Initiation of Cellular DNA Synthesis After Reovirus Infection

The synthesis of cellular DNA was measured in synchronized L cells after reovirus infection. Initiation of the synthetic phase of the cell cycle was completely inhibited in cells infected 8 h before the beginning of DNA synthesis.     

Synthesis of reovirus ribonucleic acid in L cells

Kudo, Hajime (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and A. F. Graham. Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 90:936-945. 1965.-There is no inhibition of protein or deoxyribonucleic acid (DNA) synthesis in L cells infected with reovirus until the time that new virus starts to form about 8 hr after infection. At this time, both protein synthesis and DNA synthesis commence to be inhibited. Neither the synthesis of ribosomal ribonucleic acid (RNA) nor that of the rapidly labeled RNA of the cell nucleus is inhibited before 10 hr after infection. Actinomycin at a concentration of 0.5 mug/ml does not inhibit the formation of reovirus, although higher concentrations of the antibiotic do so. Pulse-labeling experiments with uridine-C(14) carried out in the presence of 0.5 mug/ml of actinomycin show that, at 6 to 8 hr after infection, two species of virus-specific RNA begin to form and increase in quantity as time goes on. One species is sensitive to ribonuclease action and the other is very resistant. The latter RNA is probably double-stranded viral progeny RNA, and it constitutes approximately 40% of the RNA formed up to 16 hr after infection. The function of the ribonuclease-sensitive RNA is not yet known. Synthesis of both species of RNA is inhibited by 5 mug/ml of actinomycin added at early times after infection. Added 6 to 8 hr after infection, when virus-specific RNA has already commenced to form, 5 mug/ml of actinomycin no longer inhibit the formation of either species of RNA.     

Selective Irreversible Inactivation of Replicating Mengovirus by Nucleoside Analogues: a New Form of Viral Interference

We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.     

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.     

Differential sensitivity of normal and transformed human cells to reovirus infection.

Normal and simian virus 40-transformed WI-38 cells exhibited a differential sensitivity to infection with type 3 reovirus. A progressive decrease in viability began 24 to 36 h after infection of transformed cells terminating in complete lysis of cultures by 96 h. Normal cells were productively infected and continued to produce and release virus for as long as 14 days after infection, but exhibited no detectable cytopathology. Inhibition of cellular DNA synthesis began 15 to 18 h after infection in transformed cells before development of cytopathology. No inhibition of DNA synthesis was detected in infected normal cells. No significant differences were noted in the adsorption or early replication characteristics of reovirus in normal and transformed cells. Virus replication and host cell DNA synthesis in normal and transformed human cells were compared to reovirus-infected L-929 mouse fibroblast cells.     

Deoxyribonucleic Acid Synthesis in Synchronized Mammalian KB Cells Infected with Herpes Simplex Virus

We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.     

Host-Dependent Restriction of Mengovirus Replication

Mengovirus infection of a restrictive cell line, Maden's bovine kidney (MDBK), results in a virus yield 1,000-fold less than that obtained from productively infected cell lines such as L cells or Ehrlich ascites tumor cells (EAT). Cells of both types of host systems are infected with comparable efficiencies and are completely killed as a consequence of infection. Infective center assays, coupled with the observation of total cell killing, suggest that comparable numbers of cells synthesize viral antigen and release virus in both types of host system. Viral-specific ribonucleic acid (RNA) synthesis is initiated and proceeds in an identical fashion for approximately 4 hr after the infection of MDBK, EAT, or L-cells. At this time, viral RNA synthesis in MDBK ceases, whereas viral RNA synthesis in EAT and L-cells continues at a linear rate. These results indicate that none of the early viral events leading to the initiation of viral-specific RNA synthesis constitutes the primary site of mengovirus restriction in MDBK. Rather it appears that the cessation of viral RNA synthesis in restrictive cells constitutes the primary limiting event. Based on its delayed interaction with mengovirus RNA synthesis, it appears that the host-related restrictive agent is initially compartmentalized and then released as a consequence of infection subsequent to those early events in mengovirus infection leading to the initiation and continued synthesis of viral RNA.     

Evidence for a Relationship Between Equine Abortion (Herpes) Virus Deoxyribonucleic Acid Synthesis and the S Phase of the KB Cell Mitotic Cycle

Autoradiographic analyses of deoxyribonucleic acid (DNA) synthesis in randomly growing KB cell cultures infected with equine abortion virus (EAV) suggested that viral DNA synthesis was initiated only at times that coincided with the entry of noninfected control cells into the S phase of the cell cycle. Synchronized cultures of KB cells were infected at different stages of the cell cycle, and rates of synthesis of cellular and viral DNA were measured. When cells were infected at different times within the S phase, viral DNA synthesis was initiated 2 to 3 hr after infection. However, when cells in G1 and G2 were infected, the initiation of viral DNA synthesis was delayed and occurred only at times corresponding to the S phase. The times when viral DNA synthesis began were independent of the time of infection and differed by as much as 5 hr, depending on the stage of the cell cycle at which cells were infected. Viral one-step growth curves were also related to the S phase in a manner which indicated a relationship between the initiation of viral DNA synthesis and the S phase. These data support the concept that initiation of EAV DNA synthesis is dependent upon some cellular function(s) which is related to the S phase of the cell cycle.     

Mengovirus Replication in Novikoff Rat Hepatoma and Mouse L Cells: Effects on Synthesis of Host-Cell Macromolecules and Virus-specific Synthesis of Ribonucleic Acid

Novikoff cells (strain N1S1-67) and L-67 cells, a nutritional mutant of the common strain of mouse L cells which grows in the same medium as N1S1-67 cells, were infected with mengovirus under identical experimental conditions. The synthesis of host-cell ribonucleic acid (RNA) by either type of cell was not affected quantitatively or qualitatively until about 2 hr after infection, when viral RNA synthesis rapidly displaced the synthesis of cellular RNA. The rate of synthesis of protein by both types of cells continued at the same rate as in uninfected cells until about 3 hr after infection, and a disintegration of polyribosomes occurred only towards the end of the replicative cycle, between 5 and 6 hr. The time courses and extent of synthesis of single-stranded and double-stranded viral RNA and of the production of virus were very similar in both types of cells, in spite of the fact that the normal rate of RNA synthesis and the growth rate of uninfected N1S1-67 cells are about three times greater than those of L-67 cells. In both cells, the commencement of viral RNA synthesis coincided with the induction of viral RNA polymerase, as measured in cell-free extracts. Viral RNA polymerase activity disappeared from infected L-67 cells during the period of production of mature virus, but there was a secondary increase in activity in both types of cells coincidental with virus-induced disintegration of the host cells. Infected L-67 cells, however, disintegrated and released progeny virus much more slowly than N1S1-67 cells. The two strains of cells also differed in that replication of the same strain of mengovirus was markedly inhibited by treating N1S1-67 cells with actinomycin D prior to infection; the same treatment did not affect replication in L-67 cells.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.     

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.     

Fate of mRNA of L-Cells Infected with Mengovirus

Mengovirus infection of L-cells results in an inhibition of host protein synthesis which is detectable in vivo by a decreased rate of incorporation of radioactive amino acids into acid-insoluble material and by a concomitant reduction in polysome content. The inhibition of host protein synthesis occurs early in the infection cycle, at a time when there is little synthesis of viral proteins. In this paper the stability of polyadenylic acid [poly(A)]-containing mRNA of uninfected L-cells and cells infected with mengovirus is compared. Our results suggest that there is no increase in the rate of degradation of cellular mRNA upon virus infection. The continued integrity of host mRNA throughout infection was verified by acrylamide gel electrophoresis.     

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.     

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.     

DNA binding of a nonstructural reovirus protein

The specific early inhibition of DNA synthesis in reovirus-infected cells suggests that the cell nucleus is a target for virus-induced damage. We have now examined the affinity of reovirus proteins for DNA, postulating that such affinity could provide a mechanism for the inhibition. Cytoplasmic and nuclear extracts of cells labeled with [35S] methionine from 6 to 8.5 h after infection at high multiplicity was subjected to chromatography on denatured DNA - cellulose columns. Fractions from both cytoplasm and nucleus eluted with 0.6 N NaCl contained a protein with the same electrophoretic mobility of polyacrylamide slab gels as the nonstructural (NS) reovirus protein of the sigma size class. The protein also exhibited affinity for native DNA - cellulose and denatured DNA - agarose. Electrophoretic analysis is tube gels of cell extracts labeled for 48 h before infection with [14C] leucine and from 6 to 8.5 h after infection with [3H] leucine showed increased 3H label in this protein indicating it is reovirus specific. Small amounts of mu proteins also had DNA affinity. Purified virus did not bind strongly to DNA, suggesting that the binding protein is not a structural protein of the sigma size class on the outer surface of the virus. Our results provide evidence that the sigma NS protein binds to DNA. This affinity could interfere with chromosome function in the infected cell.