The influence of oxygenation on virus growth

14, 450455

VIROLOGY

The

(19Gl)

Influence

of Oxygenation

II. Effect

on the Antiviral

A. ISAAC& National

Institute

on Virus Action

J. S. PORTERFIELD, for

Medical

Research,

Accepted

April

of Interferon AND

Mill

Growth

Hill,

S. BARON1 London,

N.W.

Y

26, 1961

A good correspondence has been found between the depth to which viruses will grow in tubes filled with agar and their sensitivity to the antiviral action of interferon; viruses sensitive to interferon had a high oxygen requirement. When cultures were prepared in the presence of interferon the viruses did not grow to the same depth, showing that the action of interferon wss more pronounced in the deeper parts of the tube. Reduction in the available oxygen, either by addition of sodium thioglycolate or by increasing the depth or the concentration of agar in the overlay, caused an increase in the inhibiting titer of interferon. Increase in the available oxygen, either by incubating cultures in an atmosphere of oxygen or by using a very thin overlay, caused a decrease in the inhibitory titer of interferon. The results are consistent with the hypothesis that interferon inhibits an oxidative process which provides energy needed for viral synthesis. INTRODUCTION

An interesting finding in paper I of this series (Baron et al., 1961) was that two viruses differed in their oxygen requirements, the virus with the higher oxygen requirement having also a greater sensitivity to the antiviral action of interferon. Independently, it has been proposed (Isaacs et al., 1961) that interferon acts by uncoupling oxidative phosphorylation, possibly at a nuclear site, thus inhibiting the formation by oxidative processes of adenosine triphosphate (ATP) needed for viral synthesis. On this hypothesis, a virus that is very sensitive to interferon would have a greater requirement for ATP formed by oxidative processes than a virus that was more resistant to the antiviral

action

of interferon.

Since

the dif-

ferences in oxygen requirements of different viruses appeared to fall in line with this hypothesis, the point was studied further by investigating

the

oxygen

requirements

of

‘Senior Surgeon, United States Public Health Service, at present attached to the National Institute for Medical Research.

different viruses and by observing the effects of variations in the available oxygen on the antiviral action of interferon. The results are reported in this paper. MATERIALS

AND

METHODS

In the preceding paper (Baron et al., 1961) are described the techniques used for carrying out virus plaque assays in test-tube cultures with deep overlays or shell overlays and in petri dish cultures with slanted overlays. The method used for assaying interferon was similar to that described by Wagner (1961). Monolayers of cells in petri dishes were incubated overnight at 37” with interferon diluted in maintenance medium. Medium 199 was used for mouse embryo, monkey kidney, and human thyroid cells, which were incubated in an atmosphere of 3% CO,, and Gey’s Tris buffer containing 0.1% peptone and 0.5% lactalbumin hydrolyzate for chick embryo cells, which were then incubated in air. After 24 hours the medium was removed and the cells were infected with the appropriate challenge virus for l-2 hours. The challenge virus was next 450

INFLUENCE

OF OXYGEN

removed and an overlay of nutrient agar was added. This consisted of Eagle’s medium plus 15% skimmed milk for the monkey kidney and thyroid cells and 10% calf serum and 0.5% lactalbumin hydrolyzate in Gey’s buffered salt solution for the mouse and chick cells, Neutral red was incorporated in the overlay except in the case of mouse embryo cells, to which it was added after 48 hours’ incubation. In some experiments, cells were not pretreated with interferon but the interferon was incorporated in the overlay. Plaques were counted and the interferon titer of a preparation is taken as the dilution that reduced the plaque count to 50% of that of the controls. In the case of encephalomyocarditis virus grown in mouse embryo cells, there was great variation in plaque size and it was found that measurement of the area of the plate lysed gave a sensitive method of assaying interferon. RESULTS

Correspondence between Oxygen Requirement and Sensitivity to Interferon of Different Viruses

In the preceding paper it was noted that viruses varied in their oxygen requirements, and in the case of two viruses a correspondence was noted between sensitivity to interferon and high oxygen requirement. The relationship between sensitivity to interferon and oxygen requirement was next investigated further with other viruses which produced plaques in chick cell cultures. The results of experiments on this point are shown in Table 1, where the depth to which viruses will grow in tubes filled with agar is compared with the interferon sensitivity of the strains. Table 1 shows that in the viruses that have been investigated there has been a good correspondence between sensitivity to interferon and oxygen requirement. Efiect of Reduction of the Available Oxygen on the Antiviral Action of Interferon

The previous results have shown that differences between viruses in their oxygen requirements for growth parallel differences in their sensitivity to the antiviral action of

ON VIRUS

GROWTH.

451

II

TABLE RELATIONSHIP

AMONG

1

DIFFERENT

VIRUSES

IN

DEPTH TO WHICH THEY PRODUCE PLAQUES

Expt. no.

AND

SENSITIVITY

TO

-

INTERFERON~

-

Depth to which plaques

Virus

Sensito interferon

tl ivity

Ts

-

Bunyamwera Chikungunya Newcastle disease of fowls

12, 8 19, 14, 14 32, 26, 26

Vaccinia Fowl plague Newcastle disease of fowls

17, 17 28, 29, 29 48, 35, 44

-

l/60 l/25

l/2 l/50 l/10

l/2 -

0 The table shows (a) the depth to which viruses produce plaques in replicate chick cell cultures in tubes filled with agar. (b) the sensitivity of the same viruses to interferon as indicated by the titer of a single preparation of interferon which caused 50% reduction in the plaque count in an assay in petri dishes.

interferon. In order to see whether or not this association was fortuitous, assays of interferon were carried out under varied oxygen tensions. Three types of experiment have provided evidence that under reduced oxygen tension the antiviral action of interferon is enhanced. a. Assay of interferon in the presence of sodium thioglycolate. In preliminary experiments it was observed that the effect of adding sodium thioglycolate in a virus assay depended greatly on the depth of agar in the overlay medium. In order to take this factor into account the agar overlay was added to the plates in a slanted position so that the effect of interferon could be observed under different depths of agar. Secondary cultures of mouse embryo fibroblasts were grown for 24 hours in medium containing 10% calf serum in the presence of different amounts of interferon, infected with sufficient encephalomyocarditis virus to give 90% lysis of the cell sheet within 48 hours, and overlaid with nutrient agar medium in the slanted position. One set of interferon dilutions served as controls, and in parallel sets sodium thioglycolate in final

152

ISAACS, T.4BLE

THE

EFFECT OF S~DIIJM ANTIVIRAL ACTION

PORTERFIELD

2

THIOGLYOOLATE OF INTERFERON”

ON THE

Diameter (mm) of zone of protecSodium thioglytion at interferon dilution colate concentra- ~.~-~-...--.~~~~tion in overlay Nil l/20 l/80 l/320 control 66) 0.01 0.03 Nil control

25 26 30

5 25 15

0 25 0

0 0 0

a Secondary cultures of mouse embryo fibroblasts were incubated in 40-mm petri dishes for 24 hours with the interferon dilutions shown, They were then infected with sufficient encephalomyocarditis virus to produce 90% lysis of the cell sheet and overlaid with nutrient agar in the slanted position. In two sets of cultures sodium thioglycolate was incorporated in the agar in the dilutions shown. The readings are the diameters of the plates (measured from the greatest depth of agar) where less than 10% of cell lysis had occurred. At dilutions of l/80 and l/320 interferon in the control cultures and those containing 0.01% sodium thioglycolate, large areas of the plate showed L 10% 5 90% lysis. TABLE

3

THE EFFECT OF PARTIALLY INHIBITORY DOSES OF INTERFERON ON THE DEPTH OF VIRUS GROWTH IN AGAR TUBES INCCBATED IN AN ATMOSPHERE OF AIR OR OXYGEN~

Incubation atmosphere

Depth (mm) of virus growth at interferon dilutions shown l/10

i l/32

~ l/100

~ control Nil /

a Replicate tube cultures of chick embryo fibroblasts were infected with Chikungunya virus, and the tubes were filled with nutrient agar containing dilution of chick interferon. These were incubated either in air or in an oxygen atmosphere. The measurements give the greatest dept,h at which virus plaques were visible.

of 0.01% and 0.03% was added. After 48 hours at 37” in an incubator containing 3% CO2 , neutral red at a final concentration of l/10,000 was added and

concentrations

AND

BARON

the plaques were observed. Protection was assessed by measuring the width of the plates (reading from the greatest depth of t’he agar along the diameter) which showed less than 10% of the cell sheet lysed. The results shown in Table 2 indicate that at the higher concentration of sodium thioglycolatc the antiviral action of interferon was increased approximately sixteenfold. b. Effect of iderferon on the level at which virus growth occurs under agar. In the previous experiment it was observed that with interferon alone plaque production was most strongly inhibited where the agar was deepest in the plate. This point was tested further by setting up tubes of chick embryo fibroblasts infected with Chikungunya virus and filled with nutrient agar containing interferon in the overlay. Xft,er incubation, the depth to which virus growth occurred was measured. Line 1. of Table 3 shows that amounts of interferon which do not completely inhibit virus growth have a greater effect in the deeper parts of the tube than nearer the surface, as shown by a decrease in the depth to which virus would grow. c. Effect of depth of overlay nnd concentration of agar on the titer of interferon. A similar effect could be achieved in petri dishes by varying the volume or the concentration of agar in the overlay. Interferon assays were carried out in petri dishes containing chick embryo fibroblasts infected with approximately 250 plaque-forming units of Chikungunya virus. The overlay was varied either by increasing the volume of overlay medium or the concentration of agar in the overlay. The results of one experiment are shown in Table 4. Table 4 shows that the interferon titer was increased almost tenfold either by increasing the volume of the overlay from 3 ml to 24 ml or by increasing the concentration of agar from 1.4% to 3%, both measures which would reduce the amount of oxygen available to the cells. Effect of Increase of the Available Oxygen on the ilntiviral Action of Interferon In the experiments described above, restriction of the available oxygen led to an

INFLUENCE

OF

OXYGEN

increase in the antiviral action of interferon. The converse type of experiment was then carried out. a. Action of interferon in an oxygen atmosphere. Tube cultures of chick embryo fibroblasts were infected with Chikunguya virus and then overlaid with 6 ml agar. In some tubes interferon was incorporated in the overlay. Half the cultures were incubated in air, and the remainder were incubated in an atmosphere of oxygen. Line 2 of Table 3 shows the results of incubation in oxygen. The antiviral action of interferon which was seen when the cultures were incubated in air was less evident when cultures were incubated in oxygen since it was only seen as a very small effect at a depth of agar of more than 26 mm. In conventional plaque assays in plates with overlays of 3-4 mm depth the antiviral action of interferon would not be evident under these conditions. b. Action of interferon in monkey kidney and thyroid cells with a shell overlay. In the course of an experiment in which the action of interferon prepared in monkey and in human cells was tested in cells of both kinds, an example of the effect of increased oxygen in monkey kidney cells was observed. In this experiment the test cells were test tube cultures of monkey kidney and human thyroid cells, treated with different doses of monkey or human interferon, infected with vaccinia virus, and then overlaid with a thin shell overlay. The monkey interferon was shown in parallel tests to give a titer of about l/100 with this virus in the usual type of assay in petri dishes. However, when tested in cultures with a thin shell overlay it was found that the vaccinia virus produced large numbers of plaques within 24 hours (as described in the preceding paper) and the monkey kidney interferon was apparently of very low activity, or inactive, under these conditions. In the thyroid cells, tested at the same time, no plaques were visible until the third day, but in this case both preparations of interferon appeared to be highly active. The results are shown in Table 5. This experiment shows that monkey kidney cells behaved quite differently when

ON

VIRUS

GROWTH. TABLE

THE

EFFECT

Volume of overlay (ml)

4

OF VARYING THE OVERLAY TITER OF INTERFERON”

Concentration of agar (70)

3 6 12 24 3

453

II

Number of plaques dish at interferon

ON THE

per petri dilution

l/10

l/100

l/1000

co;;;ol

39 9 4 0 0

166 240 180 74 92

268 270 224 128 240

286 215 262 214 284

1.4 1.4 1.4 1.4 3

Q Chick embryo fibroblast cultures in petri dishes were treated with interferon in the dilutions shown, infected with approximately 250 plaque-forming units of Chikungunya virus, and overlaid with nutrient agar (a) in different volumes (b) at two different concentrations of agar. TABLE

5

ASSAYS OF INTERFERON IN MONKEY KIDNEY AND HUMAN THYROID CELLS WITH SHELL AGAR OVERLAY Number Material

tested

Monkey

kidney

of plaques Human

cells Monkey kidney interferon : l/32 l/100 l/320 l/1000 Human thyroid interferon l/32 l/100 l/320 l/1000 Controls

250 +

250 + 250 + 250 +

I

titer of interferon
4 42 I 58 255

in thyroid cells

titer of interferon >?420

:

250 +

177,200

grown with a very thin overlay than when the usual 34-mm overlay in a petri dish was used; with the thin overlay there was an increased rate of viral growth and a decreased sensitivity to interferon. It was also interesting to note the action of monkey interferon in human cells. Sutton

454

ISAACS, PORTERFIELD

and Tyrrell (1961) had previously reported that monkey interferon is active in human cells. It is not easy to deduce comparative act.ivities in the two types of cells since the titer depends on the way the assay is carried out. However, it does appear that in the human thyroid cells the monkey interferon was nearly as active as the human interferon. DISCUSSION

The results in this paper show, first, a parallelism between the sensitivity of different viruses to the antiviral action of interferon and their oxygen requirements. Secondly, it has been found that the antiviral action of interferon could be increased by restricting the available oxygen, either by adding a reducing agent such as sodium thioglycolate or by increasing the volume of agar overlay or the concentration of agar in the overlay. Conversely the antiviral action of interferon was reduced when the assays were carried out under increased oxygen tension. These findings therefore suggest that interferon inhibits an oxidative process which is presumably required for obtaining the energy, in the form of ATP, needed for viral synthesis. This suggestion would account both for the high oxygen requirement of strains that are very sensitive to interferon and for the increased antiviral action of interferon when the available oxygen is restricted. These findings fit well with the hypothesis (Isaacs et al., 1961) that interferon acts by uncoupling oxidative phosphorylation, possibly at a nuclear site. This hypothesis was put forward when it was found that interferon-treated cells showed increased glycolysis, increased uptake of oxygen, decreased uptake of inorganic phosphate, and decreased oxidat’ion of glucose via the pentose cycle-changes which resembled those produced by a known uncoupler of oxidative phosphorylation. Further evidence for the hypothesis came from the observations by Ho and Enders (1959) and Chany (1960) that cancer cells produced interferon but showed a low sensitivity to its antiviral action. According to Warburg (1956) cancer cells are not dependent on oxidative proc-

AND BARON

esses since they can produce by anaerobic processes sufficient ATP for their metabolic requirements. The same is true for early embryonic cells, and it was found that these too had very low sensitivity to the antiviral action of interferon (Isaacs and Baron, 196Oj. The implication is that cells that can bypass oxidative mechanisms should be insensitive to the antiviral action of interferon. One observation which seems to be at variance with those findings is the report by Cantell (1961) that HeLa cells are susceptible to the antiviral action of interferon, a finding which apparently conflicts with that of Ho and Enders (1959). We have studied this same point and find that the strain of HeLa cells in use in this laboratory is sensitive to the antiviral action of interferon although the sensitivity is much lower than that of normal human thyroid cells, We have also found (Raron et al., 1961) that these HeLa cells are not independent of oxygen for viral synthesis since the depth at which virus growth occurred was less than that for monkey kidney cells and increased when the tubes were incubated in an atmosphere of oxygen. It appears, therefore, that in this line of HeLa cells oxygen requirement and interferon sensitivity run parallel, but that in both respects the cells behave like noncancerous cells. At the moment it is not clear whether this could be explained by assuming that some lines of HeLa cells have reverted to the behavior characteristic of noncancerous cells, but we are investigating this further by studying other cancerous cell lines. REFERENCES

S., PORTERFIELD, J. S., and ISAACS, A. (1961). The influence of oxygenation on virus growth. I. Effect on plaque formation by different viruses. Virology 14,444-449. CANTELL, K. (1961). Production and action of interferon in HeLa cells. Arch. ges. Vimsforsch. 10,510-521. CHANY, C. (1960). lin facteur inhibiteur de la multiplication intracellulaire des virus rappelant l’interfkron, provenant de cellules cancCreuses. Le ph.&nom&e d’auto-inhibition virale. Compt. rend. acad. sci. 250,39(X-3905. Ho, M., and ENDIGRS, J. F. (1959). Further studiea BARON,

INFLUENCE

OF OXYGEN

of an inhibitor of viral activity appearing in infected cell cultures and its role in chronic viral infections. Virology 9,446477. ISAACS, A., and BARON, S. (1969). Antiviral action of interferon in embryonic cells. Lancet ii, 946 947. ISAACS,

A., KLEMPERER, H. G., and HITCHCOCK, G. (1961). Studies on the mechanism of action of interferon. Virology 13, 191-199.

ON VIRUS

GROWTH.

II

455

R. N. P., and TYRRELL, D. A. J. (1961). Some observations on interferon prepared in tissue cultures. B-it. J. Exptl. Pathol. 42,99-105. WAGNER, R. R. (1961). Biological studies of interferon. I. Suppression of cellular infection with eastern equine encephalomyelitis virus. Virology SUTTON,

13,323-337. WARBURG,

0. (1956). On respiratory in cancer cells. Science 124,267-270.

impairments