Studies on the mechanism of interferon action

VIROLOGY

22,

575-.%‘9

(1964)

Studies

on the

Mechanism

II. The Effect of Interferon Virus

on Some Early

Infection

HILTON Laboratory

of Biology of Viruses, Diseases, National institutes Accepted

of Interferon

Action

Events in Mengo

in L Cells B. LEVY

National Institute of Allergy and of Health, Bethesda 14, Maryland December

Infectious

31, 1963

The effect of interferon on early events of Mengo virus replication in suspended cultures of L cells has been studied. Interferon, at levels that almost completely block the production of Mengo virus, has no ameliorative effect on the early inhibition of normal cell protein synthesis induced by Mengo virus. Such infected interferon-treated cells go on to die in a manner similar to infected cells not treated with interferon. However, in the presence of interferon, the rapid inhibition of normal cell RNA synthesis seen in Mengo virus infection does not appear until about an hour later than usual. INTRODUCTION

While the mechanism of action of interferon is still not known, certain stages of the viral growth cycle have been eliminated as likely areas of action. The action of interferon can be stated to occur at some stage subsequent to the release of viral genome, since it is effective when infectious viral RNA is the challenge (Ho, 1961). Interferon acts prior to the assembly of the complete virus, since the synthesis of new viral RNA is inhibited (De Somer et al., 1962; Grossberg and Holland, 1962). Interferon exerts an inhibitory action on RNA metabolism in Sindbis virus-infected cells before any significant quantity of virus RNA is made (Levy et al., 1963). The difficulty with exploring this matter further relates to the fact that in most virus-host systems little is known about steps between release of infecting viral RNA and the appearance of new viral RNA. In the system of L cells supporting the growth of Mengo virus, Franklin and Baltimore (1963) have described three early even&: (1) a cut-off of normal protein synthesis, postulated to be dependent on 575

the synthesis of a new protein very early in infection; (2) a cut-off of normal RNA synthesis, postulated to be dependent upon the synthesis of another protein. [The cutoff of normal protein and RNA synthesis has been described for other virulent virus systems (Levy, 1961; Salzman et al., 1959)) but those found in Mengo virus infection occur very early, well before viral RNA is made.]; (3) the synthesis of a new RNA polymerase, presumably responsible for the synthesis of viral RNA. This paper deals with the effects of interferon on steps 1 and 2 of this system. MATERISLS

AND

METHODS

Cells. L cells, strain 929, were maintained in suspended culture in approximately logarithmic growth in Eagle’s basal medium modified for suspended culture (spinner BME) plus 5% calf serum, or as monolayers in Eagle’s basal medium (BME) and 5% calf serum. Viruses, Chikungunya virus, African strain, was kindly supplied by Dr. Alick Isaacs, Institute for Medical Reasearch, London. Mengo virus was kindly supplied

576

LEVY

by Dr. Kenneth Takemoto of this laboratory, who had obtained it from the Wistar Institute in Philadelphia. Interferon was prepared in L cells by infecting fully grown monolayers with Chikungunya virus at a multiplicity of 10. After 24 hours the whole culture was frozen and thawed, brought to pH 2 with HCl, kept at this pH at 4” for 12-18 hours to destroy residual virus, and neutralized to pH 7 with NaOH. The titer of the interferon was expressed as the reciprocal of that dilution which reduced by 50% the number of plaques obtained in L cell monolayers with a challenge of about 40 plaqueforming units (PFU) of vesicular stomatitis virus. Spent tissue culture medium was treated in the same way for use as control fluid. Radioactive metabolites. H3-Valine, 117 mc/mmole, H3-uridine, 1 curie/mmole, and H3-thymidine, 6 curie/mmole, were purchased from Nuclear Chicago Corporation. The details of their use in the experiments are given in the text and tables. Counting was done in a Nuclear Chicago windowless gas flow counter. DNA was determined by Burton’s (1956) diphenylamine procedure. RESULTS

Effect of Interferon on Cut,-off of Normal Protein Synthesis by Mengo Virus Injection

Three experiments were performed to see whether interferon prevented the cut-off of normal protein synthesis by Mengo virus, All three gave similar results. The data of one such experiment are summarized in Fig. 1. It will be seen that the inhibition of normal protein synthesis developed in what appears to be two stages. This was so for all three experiments. There was a rapid initial inhibition of about 30% during the first hour, and a subsequent further decline after 2 hours. Interferon treatment had no effect on this inhibition, nor did it affect protein synthesis in uninfected cells. The infected cells, whether treated with interferon or not, went on to die, as shown by the data of Table 1.

Interferon was effective as a virus inhibitor since the yield of Mengo virus in four experiments was reduced by 2-3 log units, from a control value of 100-300 PFU/cell. The Effect of Interferon on the Cut-off of Normal Cell RNA Synthesis by Mengo Virus Infection

The experiments were done exactly as were those described in the preceding section except that H3-uridine was used. The results of 2 of 3 experiments are summarized in Fig. 2. These experiments were carried out for only 6 hours of infection. H3-uridine incorporation after 24 hours of infection had been used as one of the tests of cell viability, as shown in Table 1. A rapid cut-off of normal cell RNA metabolism caused by the Mengo virus infection was demonstrated, and was followed by an increased synthesis of RNA, presumably viral. In the presence of interferon two differences were discernible. The later rise in radioactivity associated with the synthesis of viral RNA did not occur in the presence of interferon, indicating that interferon was inhibiting virus growth. Secondly the initial rapid cut-off of RNA synthesis was delayed by about 1 hour. Once the cut-off of RNA synthesis started in the presence of interferon, the decline in activity was continuous, and the cells went on to die. DISCUSSION

The data described in this paper show that interferon can strongly inhibit the growth of Mengo virus in L cells, but does not prevent cell death due to the virus. We have observed that interferon also inhibits the synthesis of Sindbis virus, without preventing cell destruction by the virus (Levy and Baron, unpublished observations), It would appear that at least some virulent viruses can cause cell death in spite of the decrease in viral multiplication produced by interferon. The death of the infected cells, even in the presence of interferon, may of course be due to some toxic material in the crude virus preparations or to a ‘%oxic” action of the virus

MECHANISM

OF INTERFERON I

I X-MMENGO

VIRUS

0-MMENGO

VIRUS

HOURS

,,

--I

0

I

2

AFTER 3

+

I

INTERFERON NO INTERFERON

INFECTION 4

577

ACTION

\ 5

6

”

24

FIG. 1. Effect of interferon on inhibition of normal protein synthesis induced by Mengo virus. To 500 ml of L cells (4 to 5 X 10’ cells/ml) in suspended culture in spinner BME and 5% calf serum, was added 50 ml of interferon, prepared in L cells, of original titer 160, so that the final titer in the L cell culture was 15. To another 500 ml of the same culture of L cells was added 50 ml of spent tissue culture medium, as control. Both groups of cells were incubated overnight at 37” with stirring. Each batch of cells was divided into two equal parts; the cells were centrifuged down and suspended in either 10-20 ml of BME containing Mengo virus to make a virus-cell multiplicity of 10: 1 or in the same volume of spent medium. After 20 minutes at room temperature, 150 ml of spinner BME plus 5% calf serum was added and 15-ml aliquots of each of the four cultures (infected, infected plus interferon, uninfected, uninfected plus interferon) were dispensed into small vials fitted with a small stirring bar and were incubated at 37” in a water bath to ensure rapid temperature equilibration. The time when the cells were placed at 37” was called zero time. At intervals shown in Fig. 1, 1 PC H”-valine was added to 1 sample of each of the 4 types of cultures, incubation was continued for 15 minutes more, and the cultures were rapidly chilled. The cells were centrifuged out, washed 3 times with phosphate-buffered saline (PBS), extracted twice with 10 ml of cold iO% perchloric acid, and washed once with 10 ml of alcohol; the residue was dissolved in 0.2 ml of 1 N NaOH followed by 18 ml H,O. Radioactivity, optical density at 260 rnp (OD), and DNA content were determined on suitable fractions of each sample. The specific radioactivities were calculated, using either OD or DNA content as a measure of the amount of material. The specific activities of the infected samples were expressed as percentage of the specific radioactivities of the controls. The absolute figures for specific activities were different depending on whether OD or DNA was used to measure the amount of cellular material, but the relative specific activities were not affected. itself. Other workers have also reported that viral inhibiting concentrations of interferon do not prevent the toxic action of some viruses (Gresser, 1961; Gresser and

Enders, 1962; Cantell et al., 1962). It is quite possible that the Yoxic” action seen in infected cells in the presence of interferon is attributable to a metabolic dis-

LEVY

578 TABLE

1

turbance analogous to that seenhere. Lockart (1963) has found, with Western equine encephalitis virus, that complete suppression of virus production by interferon does prevent cell death. These data show that interferon has no effect on the cut-off of normal cell protein synthesis that occurs shortly after Mengo virus infection, but does delay by about 1 hour the virus-induced cut-off of normal RNA synthesis. The decrease in RNA synthesis seenan hour late in interferon-treated infected cultures may be merely a reflection of the fact that protein synthesis had been inhibited in these cultures an hour earlier. Relevant to this are the recent experiments of Tamaoki and Mueller (1963), who showed, using puromycin, that inhibition of protein synthesis in HeLa cells can lead to later inhibition of RNA synthesis. The inhibition of RNA synthesis in interferontreated infected cultures therefore may be due to a different cause from that seen in infected cultures not treated with interferon. It is difficult to explain the difference between the effects of interferon on the Mengo

EFFECT OF INTERFERON ON METABOLIC ACTIVITY OF L CELLS INFECTED 24 HOURS WITH MENGO VIRUS Cpm incorporated into ‘ercentacid-insoluble portion age of of cells from 15 ml cell cells suspension stainbble wit1 1trypan H3Hsblue Valine t Jridine ‘I dine

Cell group

__Uninfected cells and spent medium Uninfected cells and interferon Infected cells and spent medium Infected cells and interferon

25

1525

221

4554

25

1763

237

5801

100

3

1

4

100

3

0

2

-

-

-

a Experimental details similar to those described for Fig. 1, except that 15-ml aliquots of each of the four groups of cells were incubated at 37°C for 24 hours from the time of addition of virus before exposure to 1 PC of the indicated metabolite.

I

II

I

I1 a---B

.^^

MINUTES

0

II 5060

0 FIG.

virus.

I! 8090

II

+ INTERFERON

AFTER I I50

1

I

MENGO

I

I

1 ‘300

I 360

I

INFECTION

I 180

2. Effect of interferon on inhibition of normal Details as for Fig. 1 except that the metabolite

1 240

RNA synthesis induced by was Ha-uridine, at 0.07 PC/ml.

Mengo

MECHANISM

OF INTERFERON

virus-induced inhibition of cell protein and RNA synthesis. One possibility is that interferon prevents the formation of the new protein that Franklin and Baltimore (1964) postulated as being responsible for inhibition of cell RNA synthesis, but does not prevent the formation of the one responsible for inhibition of cell protein synthesis. Another possibility is that interferon somehow interferes with the action of this hypothetical virus-induced protein even though it is made. The reported effect of interferon on RNA synthesis in Sindbis virus-infected chick embryo fibroblasts in monolayers (Levy et al., 1963), appears to be different from that just described for the Mengo virus-L cell system. In the former, the virus induced an increase in RNA synthesis 2-3 hours after infection, and interferon reduced the rate of RNA synthesis in these infected cells. It should be pointed out that the L cells in suspended culture were in logarithmic growth at the time of infection, while the CEF cells in confluent monolayers had stopped dividing. Ackermann et al. (1959) found that infection of monolayers of HeLa cells piith poliovirus caused a stimulation of RNA synthesis, while SalzmarL et al. (1959)) using the same virus in HeLa ceils groping in suspended culture, found that RNA synthesis was inhibited by infection. Possibly similar considerations apply to suspended L cells versus monolayers of CEF cells. It should be mentioned that with fully grown monolayers of L cells the Mengo virus-induced inhibition. of protein and RNA synthesis was not seen until 3 hours after infection (H. B. Levy, unpublished observations). One effect of interferon in Mengo virusinfected cells is on the events that lead to inhibition of cell RNA synthesis. The only other known very early events specifically associated with replication of the virus are the synthesis and activity of the specific viral RNA polymerase.

579

ACTION REFERENCES

W. W., LOH, P. C., and PAYNE, F. G. (1959). Studies of the biosynthesis of protein and ribonucleic acid in HeLa cells infected with poliovirus. Virology 7, 170-183. BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric determination of deoxyribonucleic acid. Biochem. J. 62,315-322. CANTELL, K., SKURSKA, Z., PAUCKER, K., and HENLE, W. (1962). Quantitative studies on viral interference in suspended L cells. II. Factors affecting interference by UV-irradiated Newcastle disease virus against vesicular stomatitis virus. Virology 17,312323. DESOIIER, P., PRINZIE, A., DENYS, P. II., JR., and SCHONNE, E. (1962). Mechanism of action of interferon. I. Relationship with viral ribonucleic acid. Virology 16,63-70. FRANKLIN, R. M., and BALTIMORE, D. (1964). Changes in RNA and protein synthesis in mammalian cells infected with a virulent virus. Symp. Fundamental cancer Res. 17th Houston 1963.. In press. GRESSER, I. (1961). Induction by Sendai’ virus of non-transmissable cytopathic changes associated with rapid and marked production of interferon. ACKERMANN,

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108,303-307.

GRESSER,I., and ENDERS,J. F. (1962). Alteration of cellular resistance to Sindbis virus in mixed cultures of human cells attributable to interferon. Virology 16,42%435. GROSSBERG,S. E., and HOLLAND, J. J. (1962). Interferon and viral nucleic acid. J. Immunol. 88, 703-714. Ho, M. (1961). Inhibition of the infectivity of poliovirus RNA by an interferon. Proc. Sot. Exptl. Biol. IJWY, H. B.

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(1961). Intracellular sites of poliovirus reproduction. Virology 15, 17%184. LEVY, H. B., SNELLBAKER, LEROY F., and BARON, SAMCEL (1963). Studies on the mechanism of action of interferon. Virology 21, 4%55. LOCKART, R. Z., JR., and HORN, B. (1963). Interaction of an interferon with L cells. J. Bact. 85, 996-1002.

SALZMAN, N. P., LOCKART, R. Z., JR., and SEBRINC, E. D. (1959). Alterations in HeLa cell metabolism resulting from poliovirus infection. Virology 9, 244-259

TAMAOKI, T., and MUELLER, G. C. (1963). Effect of puromycin on RNA synthesis in HeLa cells. Biohem.

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