The effect of phleomycin on poliovirus RNA replication

VIROLOGY

40, 441-447

The

(1970)

Effect

of

Phleomycin

on

TOBY T. HECHT2 Department

of

Microbiology Medicine,

DONALD

AND

and New

Accepted

Poliovirus

Immunology, York, New September

RNA

Replication’

F. SUMMERS3

Albert Einstein York 1046i

College

of

30, 1969

Phleomycin inhibits poliovirus-specific RNA synthesis in HeLa cells when added within the first 90 min after infection; but when it is added at later timegjin the infectious cycle, the rate of poliovirus RNA synthesis remains unchanged initially, although there is a reduction in the maximal level of RNA made. Treatment with guanidine for 1 hour prior to adding phleomycin prevents phleomycin from exerting its full inhibitory effect. The mode of action of phleomycin might be to bind to poliovirus RNA early in the infectious cycle or to prevent the synthesis of some virus-specific protein necessary for RNA replication.

bacteriophage R23 which requires RNAdirected RNA synthesis (Watanabe and August, 1968). For both 4X174 and R23, it appears that some early event in the infectious cycle of the bacteriophage is affected by the drug. This paper describes the effect of phleomycin on the replication of type 1 poliovirus, a single-stranded RNA-containing animal virus, in HeLa cells.

INTRODUCTION

Phleomycin is a partially purified, watersoluble antibiotic isolated from Streptomyces verticillus (Maeda et al., 1956) that inhibits the growth of various mouse tumors as well as Escherichia coli and HeLa cells (Tanaka et al., 1963). Early work by Tanaka and collaborators showed that the drug inhibited DNA synthesis in E. cobi and HeLa cells at concentrations at which RNA and protein synthesis were not inhibited. Subsequent in vitro studies suggest that the action of phleomycin results from the binding of the antibiotic to DNA, especially to regions rich in adenine-thymine (Falaschi and Kornberg, 1964) such that DNA synthesis is prevented. It was also found that phleomycin inhibits the replication of the singlestranded DNA bacteriophage, 4X174, presumably by interfering in some manner with the “replicative form” (Pitts and Sinsheimer, 1965), and the replication of the RNA

MATERIALS

AND

METHODS

Cell culture and infection. Suspension cultures of HeLa S3 cells were grown in Eagle’s medium (Eagle, 1959) plus 7 % horse serum at a concentration of 3-6 X lo5 cells/ml. Cells were washed twice with serum-free medium and concentrated lofold; actinomycin D was added to yield a concentration of 10 pg/ml. Unless otherwise mentioned, actinomycin D was used in all cultures in these experiments in order to turn off host cell RNA synthesis. Guanidine and cycloheximide were added to give 3 mill and 300 pg/ml, respectively, when indicated. The cells were then infected at 37” with purified type 1 poliovirus at a multiplicity of 300 plaque-forming units (PFU)/cell. Incorporation of radioactive label. Kinetics of DNA, RNA, and protein synthesis were

1 This investigation was supported by Public Health Service Research Grant No. AI-07140-04 VR and National Science Foundat,ion Grant No. GB-7187. 2 Microbiology and Immunology Departmental Training Grant No. 511 AI-00307-04. 3 Health Research Career Scientist Award No. I-456. 441

442

HECHT

AND

followed by adding radioactive precursors to the infected cell culture and then stopping incorporation at appropriate times by pipetting 0.1 ml of the culture into 2 ml of cold Earle’s solution. The samples were centrifuged, the supernatants discarded and the cells were lysed in 1 ml of water before precipitation of macromolecules with cold 5 % TCA. Assay of radioactivity into TCAinsoluble material was performed as described (Penman et al., 1964). Preparation and analysis of cell extracts. RNA from virus-infected cytoplasm was extracted by swelling the cells for 5 min at 0” in RSB (0.01 M Tris, pH 7.4; 0.01 M NaCl; 0.0015 M MgCl,), homogenizing in a tight-fitting Dounce homogenizer (Penman et al., 1963), treating the cytoplasmic extract with 1% SDS (sodium dodecyl sulfate), and heating for 1 min at 60” in order to liberate the RNA from the virus. The RNA was then sedimented through linear 15-30 % sucrose gradients prepared in NETS (0.1 M NaCl; 0.001 M EDTA; 0.01 M Tris, pH 7.4; 0.2% SDS). Whole virus, polyribosomes, and monoribosomes were analyzed by treating the cytoplasmic extracts with 1% DOC (sodium deoxycholate) and sedimenting through linear 7-47 % sucrose gradients in RSB. The contents of the gradients were analyzed for absorption at 260 rnp in a continuous-flow cell by a Gilford recording spectrophotometer. One milliliter fractions were collected, and TCA-insoluble radioactivity was determined. Chemicals. Uridine-2-14C (30 &X/cLmole), thymidine-2-14C (30-50 mCi/mmole), and uridine-5-3H (20 Ci/mmole) were purchased from New England Nuclear Corp.; RPH-14C (reconstituted protein hydrolyzate) (70-180 mCi/mmole), and RPH-3H (0.3-2.0 Ci mmole) were obtained from Schwarz BioResearch, Inc.; pancreatic ribonuclease was obtained from Calbiochem; actinomycin D was a gift from Merck and Co., Rahway, New Jersey; phleomycin, lot A9931-909, a preparation which contained 2.2 % copper, was generously supplied by Dr. I<. E. Price of Bristol Laboratories, Syracuse, New York.

SUMMERS RESULTS

The Effect of Phleomycin on HeLa Cells It was previously reported that phleomycin inhibits DNA synthesis, but not RNA or protein synthesis in HeLa cells (Tanaka et al., 1963). Since preparations of phleomycin are still impure, different samples contain different percentages of the active antibiotic. The preparation of phleomycin used in these studies gave less than 20 % inhibition of HeLa DNA, RNA, or protein synthesis at concentrations up to 100 pg/ml. The E$ect of Phleomycin fected HeLa Cells

on Poliovirus-In-

Phleomycin was added to cultures of HeLa cells at various times following infection with poliovirus. An initial 30-minute period was given to allow the virus to enter the cell and to be uncoated prior to adding the antibiotic. Figure 1 shows that phleomycin at concentration of 33 pg/ml, when added 0.5 hour after infection, gave 80 % inhibition of poliovirus RNA synthesis, and 100 pg/ml gave 95-100% inhibition. Five percent horse serum added with phleomycin 0.5 hour after infection prevented the inhibition, possibly because of phleomycin binding to serum components. One hundred micrograms of antibiotic per milliliter of infected cell culture was used in all subsequent experiments. When phleomycin was added at later times in the infectious cycle, the rate of radioactive uridine incorporation was identical to the control (Fig. 2). The initial rate of viral RNA synthesis is not inhibited when phleomycin is added 90 min after infection, although the maximum level of RNA synthesized in these cultures is less than the maximum level in control cultures (Fig. 2). Thus, phleomycin seems mainly to affect some early step in the replicative cycle, probably within the first 90 min. The Effect of Phleomycin on Infected Cells after Guanidine Treatment and Removal Cells were infected in the presence of guanidine, which allows poliovirus to attach, enter the cell, and to be uncoated, but viral

PHLEOMYCIN

INHIBITION

443

OF POLIOVIRUS

80% INHIBITION HOURS

2. Inhibition of poliovirus-specific RNA synthesis by addition of phleomycin at various times during the infectious cycle. Thirty minutes FIG.

HOURS

1. Inhibition of poliovirus-specific RNA synthesis by various concentrations of phleomycin. Phleomycin was added at, zero time to cultures infected 30 min earlier. Uridine-14C (0.15 &i/ml) was added together with phleomycin. 0-0, Control (no phleomycin); X-X, 33 rg/ml phleomycin; O-0,67 rg/ml phleomycin; A-A, 100 fig/ml phleomycin. FIG.

RNA synthesis does not proceed. Upon reversal of guanidine, the RISA replicative cycle of poliovirus is normal, approximating that of a control culture infected 30 min earlier (Summers et al., 1965). Infected cells were incubated with guanidine for 1 hour, the guanidine was reversed by washing the cells twice with cold (4”) Earle’s saline solution, and phleomycin and radioactive uridine were added. Levels of phleomycin that gave 100 % inhibition when added a 0.5 hour after infection to cells not treated with guanidine, now gave inhibition ranging from 40 to 65 %, and 200 pg/ml of phleomycin gave about 80 % inhibition under these conditions. Figure 3 shows a typical experiment where inhibition of RNA synthesis was 62 %. When the guanidine was reversed after 0.5 or 2 hours, and the phleomycin and radioactive uridine were then added, the same range (40-65 %) of inhibition was seen. Thus, although virus replication apparently

after infection (0.3 pCi/ml)

was taken as zero time. Uridine-1% was added at the time of infection. control (no phleomycin); X-X, O-0, phleomycin added 30 min after infection; O-----O, phleomycin added 90 min after infection; O---O, phleomycin added 120 min after infection; A-A, phleomycin added 150 min after infection; A-A, phleomycin added 180 min after infection.

does not occur in the presence of guanidine, its presence at the time of infection prevents phleomycin from exerting its full inhibiting effect when added at the time of guanidine reversal. When cells were infected in the presence of guanidine and phleomycin, washed after 1 hour, and radioactive uridine 100 % inhibition was obtained added, whether or not phleomycin was added again after reversal (Fig. 3). Thus, inhibition of viral RNA synthesis by phleomycin is not reversible by washing. The E$ect of Phleomycin on Infected Cells after Cyclohezimide Treatment and Removal Cells infected with poliovirus in the presence of cycloheximide show a normal virus replicative cycle following removal of cycloheximide (Penman and Summers, 1965). If, in addition, phleomycin was added at the time of infection, viral RNA synthesis was

444

HECHT

AND

SUMMERS

inhibited by 95% following removal of cycloheximide whereas synthesis was inhibited about 85 % if phleomycin was added at the time of cycloheximide removal (1 hour after infection) (Fig. 4). When both cycloheximide and guanidine were added at the time of infection and were then removed after 1 hour, and phleomycin was added together with radioactive uridine, 50-65 % inhibition of RNA synthesis was seen.

CONTROL

5

600

Kinetics of Protein Synthesis in HeLa Cells Figure 5 shows that shutoff of protein synthesis in poliovirus-infected HeLa cells 5oc

I-

84% INHIBITION

400

Reversalof Cyclohexlmids

HOURS

FIG. 4. synthesis cycloheximide was added hour after mycin); infection; moval of

0 Re,

I

I

I

1

2

3

I 4

sol of guanidine HOURS

FIG. 3. Inhibition of poliovirus-specific RNA synthesis by phleomycin added before and/or after guanidine reversal. Uridine-14C (0.125 rCi/ ml) was added in all cases at the time of guanidine reversal 1 hour after infection. O-0, control (no phleomycin); X-----X, phleomycin after guanidine reversal; O---O, phleomycin added at time of infection infection, 1 hour before guanidine reversal; A.-----A, phleomycin added at time of infection and after gltanidine reversal.

Inhibition of poliovirus-specific RNA by phleomycin added before or after reversal. Uridine-14C (0.25 &X/ml) at the time of cycloheximide removal 1 infection. O---O, control (no phleoO-----O, phleomycin added at time of X---X, phleomycin added after recycloheximide

treated with actinomycin D and guanidine occurred at a slower rate than in infected cells treated with actinomycin D and phleomycin. The rate of protein synthesis in untreated, uninfected cells declines, possibly because of lack of serum in the culture medium. Poliovirus RNA and Completed Visions Synthesized in the Presence of Phleomycin When cytoplasmic extracts of poliovirusinfected Hela cells, labelled with radioactive uridine throughout the infectious cycle, were treated with SDS and analyzed on 15-30 % sucrose gradients, a virus-specific 35 S ribonuclease-sensitive R.NA peak was seen. The 35 S RNA peak was not present when infected cells were treated with phleomycin

PHLEOMYCIN 1400

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1000

OF

POLIOVIRUS

6000

‘t PL 800 f\

1200

INHIBITION

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\

Ip. /^ ‘1 \ " 600 t\ t \\ 400 i\ \1

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2

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HOURS

FIG. 5. Shutoff of protein synthesis in HeLa cells. Cells were treated with 10 pg/ml of actinomycin D and infected with poliovirus in the presence of guanidine or phleomycin. After a 30min incubation period at 37”, 0.5-ml samples of culture were removed at 30-min intervals and added to prewarmed tubes containing 0.3 pCi of RPH-14C in a volume of 0.030 ml. From this sample a 0.01.ml background sample was immediately taken into 5% TCA and a second sample was taken 10 min later. TCA-insoluble material was filtered and counted, and the background was subtracted from the lo-min sample. Zero time is 30 min after infection. @---a, control (uninfected untreated cells) ; X-----X, cells infected in the presence of actinomycin D and guanidine; O-O, cells infected in the presence of actinomycin D and phleomycin.

30 min after infection (Fig. 6). Lower concentrations of the drug yielded 35 S material, but at reduced levels. When cells were infected in the presence of guanidine, the guanidine reversed after 1 hour (conditions which prevent phleomycin from exerting full inhibitory effect), and phleomycin added, there was a decrease in the amount of 35 S RNA compared to guanidine-treated and reversed cells without phleomycin. No accumulation of doublestranded or other RNA was observed. Infected cytoplasmic extracts were treated with DOC and analyzed for completed virions on a 747% sucrose gradient by determining the TCA-precipitable radioactivity in the 150 S region of the gradient. The amount of virus present in the cultures

FRACTION

NUMBER

FIG. 6. Analysis of cytoplasmic extracts from infected HeLa cells. 1.5 X lo7 cells infected in the presence of actinomycin D; uridine-l*C (0.3 &i/ ml) and treated 30 min after infection with phleomycin were layered onto a 15-30y0 sucrose gradient prepared in NETS at 3.5 hours after infection. Gradients were centrifuged in a Spinco SW 27 rot,or at 21,000 rpm for 17 hours at 23”. @---a, control (no phleomycin); X---X, phleomycin added at 30 min after infection; O-----O, absorbance at 260 m/.t.

treated with phleomycin after guanidine reversal is about 50% that of the control cultures not treated with phleomycin. This reduction of completed virions is proportional to the inhibition of RNA synthesis in cultures treated with phleomycin after guanidine reversal. Thus, it is concluded that the RNA made under conditions of partial inhibition is not incomplete molecules but complete viral 35 S RNA which is assembled into whole virions. DISCUSSION

The normal synthesis of poliovirus RNA in HeLa cells has been shown to have two phases: an early phase beginning within the first hour after infection during which the rate of RNA synthesis is increasing, although infectious virus is not being produced, and a later phase beginning when the number of molecules of virus RNA in the cell reaches about 4 X lo4 (at about

446

HECHT

AND

2 hours depending upon the multiplicity of infection), during which the rate of RNA sythesis is constant and mature virus particles are found (Baltimore et al., 1966). From the results shown in Fig. 2, it is concluded that phleomycin acts upon virus replication mainly during the early phase of RNA synthesis, since addition of the drug to infected cultures 90 min or more after infection results in little inhibition of the initial rate of RNA synthesis. This is in contrast to the action of phleomycin on the RNA-bacteriophage R23 where RNA synthesis is stopped within minutes after addition of the antibiotic at all times during the infectious cycle (Watanabe and August, 1968). The DNA-bacteriophage 4X174, like poliovirus, seems to respond to phelomycin only during the early phase of nucleic acid replication (Pitts and Sinsheimer, 1965). Guanidine probably does not prevent the uncoating of poliovirus in HeLa cells (Summers et al., 1965), yet addition of phleomycin at the time of guanidine reversal to cells that were infected in the presence of guanidine still results in an inhibition of RNA synthesis. Furthermore, the same range of inhibition was found to occur if the infected cells remained in guanidine for a 0.5, 1, or 2 hours after reversal. Thus, the RNA synthesis itself is being affected by phleomycin, and probably not the uncoating of the virus. The reason that phleomycin does not completely inhibit RNA synthesis after infection in the presence of guanidine is not clear, although it is possible that some virusspecific RNA polymerase is synthesized from the input virus RNA molecules during guanidine repression. After reversal of guanidine, these polymerase molecules might then be able to function despite the presence of phleomycin. However, cells treated with phleomycin after infection in the presence of guanidine and cycloheximide, when presumably no virus-specific proteins are made, show the same 40-65% inhibition of viral RNA synthesis as when infected cells are first treated with only guanidine. This result seems to rule out an explanation of the decreased inhibitory effects of phleomycin in

SUMMERS

infected cells pretreated with guanidine based on the synthesis of virus RNA polymerase, unless guanidine somehow blocks the effect of cycloheximide and thereby permits some polymerase to be synthesized. The fact that cells infected in the presence of cycloheximide alone, washed after 1 hour, and phleomycin added show a somewhat greater inhibition of virus RNA synthesis, supports the idea that guanidine may interfere with cycloheximide. Phleomycin may inhibit poliovirus RNA synthesis by blocking synthesis of the virusspecific polymerase. The fact that the drug exerts a greater effect when added early in the infectious cycle may only reflect a larger pool of active polymerase molecules available later in the cycle. It has been shown that phleomycin can bind to DNA in vitro (Falaschi and Kornberg, 1964) and to the RNA of reovirus (Watanabe and August, 1968). Similarly, phleomycin might bind to poliovirus RNA and, for example, inhibit the RNA polymerase from functioning. However, phleomycin does not inhibit the poliovirus RNA polymerase complex from synthesizing RNA in vitro (Ehrenfeld, personal communication), but de novo association of polymerase with template RNA may not occur in the in vitro reaction, and so the possibility cannot be excluded that phleomycin inhibits the initial steps of polymerase-template interaction. REFERENCES BALTIMORE, (1966).

D.,

GIRARD,

M.,

and

DARNELL,

J. E.

Aspects of the synthesis of poliovirus RNA and the formation of virus particles. Virol-

ogy 29, 179-189. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. FALASCHI, A., and KORNBERQ, A. (1964). Phleomy&, an inhibitor of DNA polymerase. Federation Proe. 23, 940-945. MAEDA, K., KOSADA, H., YAGISHITA, K., and TIMEZAWA, H. (1956). A new antibiotic phelomycin. J. Antibiotics (Tokyo) Ser. A 9, 82-85. PENMAN, S., and SUMMERS, D. F. (1965). Effects on host cell metabolism following synchronous infection with poliovirus. Virology 27, 614-620.

PHLEOMYCIN

INHIBITION

S., SCHERRER, K., BECKER, Y., and DARJ. E. (1963). Polyribosomes in normal and poliovirus-infected HeLa cells and their relationship to messenger RNA. Proc. Natl. Acad.

PENMAN, NELL,

Sci. U.S. 49,654-662. PENMAN, S., BECKER,

Y., and DARNELL, J. E. (1964). A cytoplasmic structure involved in the synthesis and assembly of poliovirus components. J. Mol. Biol. 8, 541-555. PITTS, J., and SINSHEIMER, R. L. (1965). Effect of phleomycin upon replication of bacteriophage +X174. J. Mol. Biol. 15, 766-680.

OF POLIOVIRUS

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D. F., MAIZEL, J. V., and DARNELL, J. E. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U.S. 54, 505-513. TANAKA, N., YAMAGUCHI, H., and UMEZAWA, H. (1963). Mechanism of action of phleomycin, a tumor-inhibitory antibiotic. Biochem. Biophys. Res. Commun. 10, 171-173. WATANABE, M., and AUGUST, J. I. (1968). Replication of RNA bacteriophage R23. II. Inhibition of phage-specific RNA synthesis by phleomycin. SUMMERS,

J. Mol.

Biol.

33,21-33.