VIROLQOY 69, 727-736 (1976)

Interferon-Sensitive I. Isolation

Mutants

and Biological

of Mengovirus

Characterization’

EDWARD H. SIMON, S. KUNG, T. T. KOH,2 AND I’. BRANDMAN Department

of Biological

Sciences, Purdue

University,

West Lafayette,

Indiana

47907

Accepted September 22,1975 Three interferon-sensitive (is) mutants of mengovirus have been isolated and the properties of one (is-l) studied in detail. This mutant, is-l, grows normally in control L cells, but in cells pretreated with interferon, its yield is at least lo-fold lower than wild type (is+). Furthermore, is-l attains 95% of its final yield 12 hr postinfection whether in the presence or absence of interferon, whereas in protected cells, is+ does not reach this level until 20 hr postinfection. In mixed infection in the presence of interferon, is+ can rescue is-l, implying that the mutation affects a diffusible product. Most cells protected with 10 units of interferon and infected with is-l survive the infection and are able to form clones, as measured by colony-forming ability. If protected cells are treated with actinomycin D immediately following infection, the yields from cells infected with is-l are enhanced and the yields from cells infected with is+ are lowered, the net result being complete reversal of the is-l phenotype. These results are consistent with the hypothesis that the is-l function protects the virus genome from an induced cellular nuclease. 1NTRODUCTION

While it is well known that different classes of viruses have different sensitivities to interferon, the reasons for this are unclear. Occasionally this difference is even exhibited between viruses in the same class, such as simian virus 40 (SV40) and polyoma (Oxman, 1973) and, in a few cases, different strains of the same virus have different sensitivities (Lomniczi, 1973). These facts, combined with the observations of Fleischmann and Simon (1973) and Chang et al. (1973) that the primary effect of interferon is to induce a delay in virus synthesis, led us to believe that viruses have an active mechanism to overcome the inhibition induced by interferon. This implied that single-step interferon-sensitive (is) mutants could be isolated, and a screening program to isolate such mutants was devised. The program was successful and three mutants have ’ Supported by Grant No. AI 11270 from the National Institutes of Health. * David Ross Predoctoral Fellow of the Purdue Research Foundation.

been isolated. This paper will discuss the biological characterization of one of these and suggest a possible mode of action for it. MATERIALS

METHODS

Cells and uirus. Mouse L cell (strain 929) cultures were maintained at 37” in a humidified, 5% CO, atmosphere in Eagle’s minimal essential medium (MEM) supplemented with 10% newborn calf serum (growth medium). Cells were routinely passaged every 4-7 days. Mengovirus, originally supplied by R. Franklin, was selected for resistance to heat decay at 37”, plaque purified, and grown in L cell monolayers on 150-mm glass petri plates in 30 ml of growth medium. After 18-24 hr, the monolayers were freeze-thawed three times. The medium was collected, spun at low speed to remove coarse cell debris, distributed in l-ml aliquots, and stored at -60”. Titers ranged from 2 x lo7 to 2 x lO*/ml. Plaque assay. The plaque assay system used a starch overlay and was described in detail by Fleischmann and Simon (1973).

727 Copyright 6 1976by Academic Press, Inc. All rights of reproduction in any form reserved.

AND

728

SIMON

Znterferon preparation and titration. Interferon was induced by infecting L cells with Newcastle disease virus (NDV) which in turn was inactivated by dialysis at pH 2 for 5 days. Details are given in Fleischmann and Simon (1974). Purified interferon (6 x lo6 NIH mouse reference standard units/mg of protein) was kindly supplied by Dr. P. Lengyel. Isolation of interferon-sensitive (is) mutants of mengovirus. Mengovirus was mutagenized by treatment with nitrous acid (HNO,) as described by Granoff (1961). One milliliter each of 4 M sodium nitrite and 1 M sodium acetate, pH 4.8, was added to 2 ml of stock mengovirus. After a 5-min treatment at room temperature, an O.i-ml aliquot of the reaction mixture was added to 9.9 ml of cold phosphate buffer to stop the reaction. The mixture was then titered and showed a survival of 10p3. Individual plaques were picked into 0.5 ml of MEM; preliminary studies showed that each plaque yielded about 5000 plaque-forming units (PFU). The sensitivity of the viruses in each plaque to interferon was tested as follows: 5 x lo5 L cells were seeded in each small cup of a 96 CV-TL multidish plastic tray (Linbro). About 80 PFU from each plaque were placed on two cups, one protected with 1 unit of interferon, the other not. The cups were then overlaid with 0.5 ml of starch as described by Chang et al. (1974) and the resulting plaques counted 24 hr later. The inhibition produced by 1 unit of interferon was calculated for each plaque. Virus from the five most sensitive plaques were grown into stocks and tested further as described in the text. Growth curves. 4 x 10” L cells were seeded onto 60-mm tissue culture plates. The plates were overlaid with 2 ml of varying amounts of interferon diluted in MEM + 10% calf serum. Twelve hours later, the fluid was removed and the plates were washed and infected with is-l or is’ at a multiplicity of 10 PFU/cell. After a 45-min adsorption period, the plates were washed three times and 2 ml of growth medium was added to each plate. Extra- and intracellular virus titers were determined at various times postinfection. Extracellular virus: O.l-ml aliquots of growth medium

ET AL

were taken and diluted with 0.9 ml of fresh growth medium, and the plates were then returned to the incubator. Intracellular virus: growth medium was removed from the plates being tested, and the plates were washed three times. Two milliliters of fresh medium was then added to each plate, the cells lysed by one freeze-thaw cycle, and the supernatant fluid sampled. All samples were stored at -20” until assayed, usually within 24 hr. Cloning experiments. L cells were protected with various concentrations of interferon and infected as described above. Twenty-four hours postinfection, the plates were observed under the microscope and the supernatant fluid sampled for virus. After washing with phosphate buffer, the cells were trypsinized, and the number of intact cells was determined by counting them in a hemacytometer. There was rarely any difficulty in distinguishing an intact cell from a lysed one. To determine growth rates, the intact cells were diluted in growth medium and seeded onto 35-mm tissue culture plates at 2 X lo4 cells in 2 ml of medium per plate. Half of the plates were protected with 1 unit of interferon, and the other half were left unprotected. Three areas were marked on each plate, and the cells in each marked area were counted every day. Cloning efficiency was determined by plating into tissue culture plates a suitable number of intact cells in growth medium supplemented with 20 pg/ml of serine. In some experiments, half of the plates contained 1 unit of interferon to prevent spread of infection by residual virus. Clones were counted 7 days later. Genetic studies. Cells were infected as previously described with is+, is-l or a mixture of is-l and is+. Twenty-four hours postinfection the supernatant fluids were titered. Individual plaques derived from unprotected cells infected with is+ or is-l and from cells protected with 2.5 units of interferon and infected with is+ x is-l were picked and the total virus distributed into two cups of the multidish Linbro tray, each containing a monolayer of L cells. In each case, one cup had been overlaid with 2 units of interferon for the previous 12 hr while the other was a control. The cups

INTERFERON-SENSITIVE

729

MUTANTS

were checked daily. Both protected and control cells were destroyed by is+ within 2-3 days, while is-l destroyed only the control cells. Studies with antimetabolites. Cycloheximiok. L cell monolayers were infected with either is+ or is-l overlaid with 2 ml of

growth medium containing 50 a/ml of cycloheximide. At suitable times, the cycloheximide-containing medium was removed and replaced with standard medium. In each case, 24 hr following replacement the supernatant fluid was sampled for infectious virus. In one experiment, described in the text, the efficiency of protein inhibition was monitored by adding radioactive amino acids. Actinomycin D. Actinomycin D at 5 pg/ml was added to L cells, some of which had been pretreated with interferon, at various times following infection. In all cases, the cells were infected at a multiplicity of 5-10 PFU/cell. RESULTS

Isolation

of is Mutants

Plaques formed by mutagenized viruses were tested as described in Materials and Methods, and the amount of plaque inhibition induced by 1 unit of interferon was calculated for each isolate by comparing the number of plaques found on control and interferon-treated cups. The data are presented in Fig. 1. In the initial isolation, 57 of the 107 mutagenized plaques showed 70% or more inhibition, and, upon retesting, most of them retained their relative interferon sensitivity. Five of the most sensitive plaques were grown into stocks and tested for their ability to grow in interferon-protected L cells. One of these, is-l, proved to be markedly sensitive to interferon (see Fig. 2A). In the course of these experiments, a presumptive interferon-resistant mutant was also isolated. In the presence of interferon, it consistently gave yields lo-50% higher than wild type. No further work has been done with this mutant. Sensitivity

to Interferon

The interferon sensitivity of a virus is conventionally measured by a plaque inhibition test; by this criterion, is-l was only

% lNHlBlTlON

Frc. 1. Sensitivity of mutagenized mengovirus. One hundred and seven plaques were picked and virus from them tested for interferon sensitivity by a plaque inhibition test; 57 of the most sensitive plaques from the first screen were then retested. Details are given in Materials and Methods. (01, Control; (al, first test; (ml, second test.

1.5 times as sensitive as is+, that is, an interferon preparation that titered 100 units/ml using is+ as the test virus titered 155)units/ml against is-l. Sensitivity can also be measured by studying the effect of interferon on virus yield. The results of two experiments are presented in Fig. 2 and show that is-l is 510 times more sensitive than is+ in terms of the amount of interferon needed to reduce the final yield to comparable levels. Figure 2A shows the time course of infection for is+ and is-l on cells pretreated with different amounts of interferon; in Fig. 2B, the 24-hr yield from cells pretreated with the indicated levels of interferon is presented as a percentage of the control virus yield. Note that in the absence of interferon, is+ and is-l gave equal yields. The fact that is-l did not accumulate in interferon-treated cells was demonstrated by an experiment parallel to the one in Fig. 2A. It showed that disrupting the cells by three freeze-thaw cycles to release intracellular virus did not affect the results. While variations in yields of virus from interferon-treated cells were

730

SIMON

I

1°k7i+e4 TIME

IN HOURS

I

‘\.

I

’ ’ -I d

2 4 8 UNITS Cf= IN-ML

16

32

FIG. 2. (A), Growth of is+ and is-l in the presence and absence of interferon. Fresh L cell monolayers were prepared, and 6 hr after trypsinization they were treated for 12 hr with interferon koncentrations shown in parentheses) and infected at an m.o.i. of 5-10 PFUkell. (B), Relative yields of is+ and is-l as a function of interferon concentration. Monolayers were treated as in (A) and sampled for virus 24 hr postinfection.

common, the ratio of titers obtained from cells infected with is+ and is-l was relatively constant, as shown in Fig. 2B. Fleischmann and Simon (1973) and Chang et al. (1973) used studies of isolated single cells to show that a major effect of low doses of interferon was to delay the onset of mengovirus synthesis. Since the amount of delay varied from cell to cell, the net effect was a slow rise in virus titer from protected cells, as shown in Fig. 2A. In order to compare the kinetics of production of the two viruses, the data of Fig. 2A was plotted as the fraction of final yield (Fig. 3). The data show that, although is-l (0.5) and is+ (2.5) are equally sensitive to interferon in terms of final yield, unlike the wild type, the mutant shows little or no delay. This observation is highly reproducible. Even at greater levels of interferon (see Fig. 6), the mutant shows little delay in virus production, and whatever virus is formed is released at the same time as the wild type. Genetic Studies

The ability of is-l and is+ to grow in each other’s presence was determined by assay-

ET AL.

ing the yield from a mixed infection in cells previously protected with interferon. If is-l were dominant, the yield of is+ would be reduced to is-l levels, and, if recessive, both would grow to wild type levels. A third possibility was that each virus would grow independently of the other. This would be expected if the is-l genome had a structural defect which rendered it susceptible to host defenses. The results of the experiments are shown in Table 1. Plaques were picked and then genotypes determined as described in Materials and Methods. The results show that is-l is recessive to is+ and suggest that is-l is deficient in some diffusible product that can be supplied by the wild-type virus. Cell Survival

It has long been noted that, except in the presence of massive amounts of interferon (Haase et al., 1969), cells do not survive infection even though the virus titer is reduced by several logs, and their cytopathic effect is greatly inhibited. The data of Fig. 4 show that, even in the presence of low concentrations of interferon, a considI

I

Time

1

I

I

16

20

24

(hr)

FIG. 3. Kinetics of virus growth. The data of Fig. 2A are plotted as fraction of final yield to emphasize the different rates at which is-l and is+ are produced in the presence of interferon.

INTERFERON-SENSITIVE TABLE RECOVERY

Virus

OF is-l

1

AND WILD INFECTION’

IF Fraction of con(units) trol yield Expt l*Expt

is+ is-l is+ X is-l -

TYPE

0 2.5 0 2.5 0 2.5

1 0.67 1 0.1 1 0.9

2b

1 0.46 1 0.022 1 0.18

FROM MIXED

is-l’

(%)

Expt 1 Expt 2 4(24) 92(24) 39(69)

-

41(37)

36(69)

’ All cells were infected with a total m.o.i. of 10 PFU/cell and samples taken 24 hr after infection. The genotype of the resulting viruses was determined as described in Materials and Methods. b Control titers averaged 3 x 10’ and 3.4 x 10s, respectively. ’ Total plaques analyzed are given in parentheses, the values for is+ and is-l verify the accuracy of the assay.

FIG. 4. Survival of infected cells. Cells were protected with interferon and infected with virus as described in Fig. 2A. Twenty-four hours after infection, intact cells were determined by hemacytometer counts and surviving cells by cloning. Details are given in Materials and Methods.

MUTANTS

731

erable number of the cells infected with either is+ or is-l remains intact. Even more striking is the fact that a large proportion of these can form clones. The observation that substantial numbers of is+-infected L cells survive the infection has been made several times with two different L cell lines. However, in other experiments, few, if any, cells survived the is+ infection. The source of this variability is unknown. Nevertheless in every experiment the basic differences between infection with is+ and is-l were still apparent: Except at very high levels of interferon, cultures protected with is-l contained more intact cells, more surviving cells, and produced a lower yield of virus. In most cases, the differences observed were at least a factor of 10. Since these experiments were performed with an unpurified preparation of interferon, it was conceivable that a substance other than interferon was responsible for the protection. However, they have been confirmed by using a purified preparation obtained from P. Lengyel. Unprotected cultures contained no clone-forming cells after infection. To confirm that this was not due to superinfection of surviving cells by residual virus, uninfected cells were added to the intact cells remaining after infection with is-l or is+. The results are given in Table 2 and show that residual virus had little, if any, effect on cell survival. Clones were usually counted after the cells had undergone from five to seven divisions. The data of Table 3 show that the antiviral activity induced by low levels of interferon is lost after three divisions. Hence, if an intact is-l genome were present in surviving cells, it would be able to resume growth and destroy the clones. The absence of this effect suggested that the is1 genome had been destroyed. A further series of experiments have been performed to confirm that cells surviving 24 hr after infection with is-l were unable to yield virus. (i) In an experiment similar to that presented in Fig. 3, the surviving infected cells were cloned in normal medium or in medium supplemented with 1 unit/ml of interferon, which was sufficient to sup-

732

SIMON ET AL,. TABLE 2 CLQNE FORMATION IN THE PRESENCE OF MENGOVIRUS~

Virus

(m&ml)

Intact cells seeded (No.)

Uninfected cells seeded

Clones (No.)

Clones (av No.)

0 0 0 0 100 100

0 1 0, 0 95, 159 170, 193 81, 40 59, 70 45, 58

0 1 0 127 181.5 60.6 64.5 51.5

Residual viruses (PFU/ml) 1st day

7th day

(No.)

is+

-

is-l is+

10 10 10 -

is-l

is-l -

2 2.2 8 1.3 1.3

x x x x x -

103 103 103 103 103

100

100

6 5.1 8.3 >5 4

x x x x x 9x 9.5 x

lo4 106 103 103 103

3.8 x 102 9.5 x 102 3.6 x lo2 40 4.5 x 10s