Conditional lethal mutants of rabbitpox virus

VIROLOGY

a8,

i%2--5%l

(1966)

Conditional I. Isolation

Lethal

of Host Cell-Dependent

J. F. SAMBROOK, Department

Mutants

of Rabbitpox

Virus

and Temperature-Dependent

B. L. PADGETT,’

AND

J. K. N. TOMKINS

The John Curtin School of Medical Research, Australian Canberra, A.C.T., Australia

of Microbiology,

Accepted November

Mutants

National

University,

23, 1965

Growth of a cloned stock of rabbitpox virus in the presence of the mutagens 5bromodeoxyuridine and 2-aminopurine resulted in a reduced yield of infectious virus. Temperatureand host-dependent mutants could be detected in these yields. Methods were devised for the large-scale isolation of both types of conditional lethal mutants and for enriching the vield of temnerature-sensitive mutants by the use of the viral inhibitor isatin 8-thiosemicarbazone. -

”

INTRODUCTION

Most earlier investigations of recombination with animal viruses have utilized nonlethal mutants with easily recognized novel phenotypes (e.g., neurovirulence and serological character for influenza viruses; plaque and pock character for poxviruses, Newcastle disease virus, and polioviruses). The yield of useful information from this work has been limited, and it is apparent that a new approach is necessary if the genome of representative animal viruses is to be adequately characterized (Fenner and Sambrook, 1964). Conditional lethal mutants, used by Edgar and Epstein for mapping bacteriophage T4 (Epstein et al., 1963) may constitute this new approach, which is already being exploited by Cooper (1965) with poliovirus and Burge and Pfefferkorn (1965) with Sindbis virus. With poxviruses, studies by McClain (1965) and McClain and Greenland (1965) suggested that mutants comparable with Epstein’s amber mutants might be obtained from rabbitpox virus, using PK-2a or L929 cells as the nonpermissive host, and on general principles it seemed probable that temperaturesensitive (ts) mutants could also be obtained. 1 Special Research Fellow, Health Service, 1964-1966.

United

States Public 592

This paper describes the selection methods used to obtain such mutants. The mutagens used were 5-bromodeoxyuridine, which is known to be incorporated into poxvirus DNA with high efficiency (Easterbrook and Davern, 1963), and 2-aminopurine. The mutants isolated are conditionally lethal in that they differ from wild-type rabbitpox (RP+) virus in one of two ways. Either they fail to multiply at 39.5” (ts mutants) or they fail to multiply in PK-2a cells (p mutants). Both sets of mutants multiply in chick embryo fibroblasts (CEF) at 34.5’. MATERIALS

AND

METHODS

Cell culture. The routine maintenance of PK-2a cells (a continuous line of adult swine kidney cells) and the preparation of PK-2a and CEF monolayers have been described (McClain, 1965). PK-2a “minilayers” were prepared in WHO hemagglutination (HA) trays, by seeding each of the eighty roundbottomed cups with 2 X lo5 cells in 0.5 ml growth medium. Confluent monolayers were formed in the cups after overnight incubation at 37”. Media. Growth medium consisted of 5% calf serum (CEF) or 5% foetal calf serum (PK-2a) in Hanks’ solution with 0.5% lactalbumin hydrolyzate and 0.05% bicarbonate. Maintenance medium consisted

ISOLATION

OF MUTANTS

of 0.5 % lactalbumin hydrolyzate, 0.1% yeast ext’ract, and 0.1% bovine albumin in Earle’s salt solution with 0.1% bicarbonate. Virus infectivity a~ays. Plaque assays were based upon the methods of McClain (1965). Confluent monolayers were grown in 50-mm glass petri dishes, the virus inoculum of 0.1 ml was allowed to adsorb for 2 hours at room temperature, and t’he overlay consisted of maintenance medium containing 1% agar. Cultures were incubated for 3-4 days in 5 % COZ in air (at 34.5” unless ot’herwise stated). The monolayers were then stained with neutral red. Cloning of virus. The parent virus was wild-type rabbitpox, RP+ (RP-Utrecht; Fenner, 1958), of which a cloned stock was prepared as follows. A single-pock suspension of RP+, diluted to produce about 5 plaques per dish, was inoculated onto PK-2a monolayers. After 4 days’ incubation at 39.5” a single well-isolated plaque was picked by aspiration of cell debris through the agar layer into 1 ml of maintenance medium. This plaque suspension was frozen and thawed once, t,reated for 90 seconds with a Mullard ultrasonic drill, and then serially passed twice in PK-2a monolayers under liquid medium in Roux bottles at 39.5”. The virus so obtained had a titre of 3 X 107 PFU/ml when titrated on PK-2a monolayers at 34.5”, and this stock was used in all experiments described. Temperature control. Cultures incubated at 39.5” were sealed in plastic boxes and immersed in a water bath regulated at 39.5 f 0.05” by means of a Braun (Melsungen, Germany) Thermomix II control unit. Cultures were incubated at 34.5’ in a standard incubator. Chemicak. 5-Bromodeoxyuridine (BDU) and 2-aminopurine (AP) were purchased from the California Corporation for Biochemical Research, Los Angeles. Isatin @thiosemicarbazone (IBT) was obtained from Dr. K. Easterbrook. Hemagglutination trays. WHO hemagglutination (HA) trays were cleaned by immersion in 4 % caustic soda for several hours, followed by numerous rinses with distilled water. No further sterilization was attempted, and contamination was not a problem.

OF RABBITPOX

593

VIRUS TABLE

1

YIELD OF VIRUS FROM CEF INFECTED WITH OF MUTAGENS, AND CLONED RP+ IN PRESENCE THE PERCENTAGES OF ts MUTANTS IN THE YIELDS 5 Bromodeoxyuridine

(BDU)

T

2 Aminopurine

(AP)

COllCt?~-

tration of mutagen CudmU

Yield bm PFU)

0 1 1.5 2 3 4 5 6 7 9

6.45 5.94 5.80 5.43 4.70 4.32 3.90 3.23 2.69 2.30

COIlCell F‘er cent I.ration <)f ts 1 mutage1 nnutants bdml) -


-

IS

nmtants -/-

0 100 -

6.45 6.07 -

300 400 500 -

6.00 5.78 5.74 -

700 900

5.45 5.00 -

P‘3 cent

Yield gfjg


1.2 1.2 3.8

-I

-

a CEF monolayers in small screw-capped

bottles were inoculated with lo* PFU of cloned RP+. After adsorption for 2 hours at room temperature the inocula were removed and the cell sheets were washed twice. One millilitre of maintenance medium containing BDU or AP was then added, and the monolayers were incubated at 34.5”. After 42 hours’ incubation, the cell sheets were washed and the virus yield was titrated on CEF at 34.5”. RESULTS

Growth of RP in the Presence of Mutagens CEF monolayers (2 X lo6 cells) in small screw-capped bottles were inoculated with lo4 PFU of cloned RP+. After adsorption (2 hours at room temperature), the inocula were removed, and the cell sheets were washed twice. One millilitre of maintenance medium containing BDU or AP was then added, the bottles were sealed, and the monolayers were incubated at 34.5”. After 42 hours’ incubation, the mutagen was removed, the cell sheets were washed once with maintenance medium, and finally 1 ml of mutagen-free maintenance medium was added. The monolayers were subjected to one cycle of freezing and thawing and 15 seconds’ sonication before the virus yield was titrated on CEF monolayers at 34.5”. The results are shown in Table 1. It is seen that the more mutagen present during the

594

SAMBROOK,

PADGETT,

growth cycle, the less the yield of viable virus. Isolation

of Conditional

Lethal Mutants

Techniques. The techniques employed involved the growth and isolation of clones of virus with selection pressure against mutants of the desired type as low as possible, followed by characterization of the isolated clones with selection pressure on, i.e., under restrictive conditions. (a) Isolation of clones. A dilution of the virus preparation under test was used which was known to give about lo-20 plaques on CEF after incubation at 34.5”. At this temperature and in these cells both sorts of mutants formed plaques, which were usually indistinguishable in appearance from those of RP+. Well-separated plaques were picked by aspiration of cell debris through the agar overlay, using a different capillary pipette for each plaque. The isolates were

AND

TOMKINS

stored at -10” as suspensions in 0.8 ml maintenance medium in the cups of labelled HA trays. (b) Testing of isolates. The plaque isolates were tested for their ability to grow on PK-2a minilayers at 39.5’ and 34.5’. The medium was aspirated from the minilayers, and 0.1-0.2 ml of each plaque isolate was inoculated onto a PK-2a minilayer on each of two separate HA trays so arranged that the original isolate and the two PK-2a minilayers infected with it were in identical positions on their HA trays. A different pipette was used to handle each plaque isolate. After all the minilayers in a single HA tray had been inoculated, the virus was allowed to adsorb for 1 hour at room temperature, and then 0.6 ml of maintenance medium was added to each cup. One of the homologous pair of infected HA trays was incubated at 34.5” whilst the other was

FIG. 1. Isolation of ts and p mutants. PK-2a minilayers, prepared as described in Methods, were infected with 0.1 ml of plaque isolates obtained from RP+ grown in the presence of 5-bromodeoxyuridine. After incubation for 65 hours the minilayers were stained with crystal violet. The photograph shows portions of two hemagglutinin trays infected with the same plaque isolates and incubated at 34.5” (on the left) or 39.5”. Most of the isolates produce plaques at both high and low temperatures, although the plaques at high temperature are always smaller. One isolate (circled) failed to show plaques at high temperature although there were plaques formed at low temperature: this isolate is a presumptive ts mutant. Two isolates failed to form plaques at both high and low temperature: these are presumptive p mutants.

ISOLATION

OF MUTANTS

OF RABBITPOX

TABLE

~~~

595

VIRUS

2

POSSIBLE RESULTS OF TESTS OF POTENTIAL MUTANTS Low TEMPERATURES”

IN PK-2a

CELLS AT HIGH

AND

Class

34.5”

39.5”

Conclusion

1 2 3 4

Plaques Plaques No plaques No plaques

Plaques No plaques Plaques No plaques

Not ts or p mutant Possible ts mutant Thermoefficient mutant Possible p mutant

a Plaques were picked from RP+ virus grown in the presence of BDU or AP. Virus from plaques was then tested on PK-2a monolayers at 34.5” and 39.5” (see text).

incubated at 39.5”. After 65-67 hours’ incubation (i.e., after all primary but before any secondary plaques had developed) both sets of minilayers were stained with a 1% solution of crystal violet. After staining, the plaques stood out clearly as holes in the intense blue of the cell sheet. The plaques in the minilayers incubated at 34.5” were larger in size than those in minilayers incubated at 39.5” (Fig. 1). Up to 40 plaques per minilayer could be counted with ease; above this number the plaques tended to fuse together. With RP+ t,he sensitivity of the minilayers as an assay system is 0.6 that of PK-2a monolayers. The four possible results that are obtainable from the testing of a single isolate are shown in Table 2. In fact, class 3 was never found although such thermoefficient mutants have been isolated from other viruses (Inoue and Kate, 1963; C. I. Davern, personal communication; P. D. Cooper, personal communication). Results. The stock of RP+ used in these experiments had been cloned and grown in PK-2a cells at 39.5“ and would therefore be expected to contain very few ts or p mutant’s before being treated with the mutagens. This virus was inoculated onto CEF monolayers at 34.5” and 1600 clones were picked. All these isolates fell into category 1 of Table 2, demonstrating that the stock of parental virus was essentially free of ts and p mutants. The virus was then grown in CEF at 34.5” in the presence of varying concentrations of BDU or AP, and 80 plaques were picked from each of the virus yields obtained. The proportion of isolates that fell into category 2 (ts mutants) was determined for each concentration of muta-

gen used. The results are shown in Table 1. The number of ts mutants found was proportional to the concentration of mutagen used, and, since there are absolute increases in their frequency, the selection of preexisting mutants from the wild-type stock is extremely unlikely. Large-scale isolation of ts and p mutants was undertaken using a virus stock grown in CEF at 34.5” in the presence of 1.5 pg BDU per millilitre. Several thousand plaques have been isolated from this stock and, on testing, most of the isolates fell into category 1 of Table 2. A small proportion, however, fell into categories 2 and 4. These presumptive mutants were recovered from the storage plates and passed at 34.5” in CEF. This usually resulted in a virus stock with a titre of about lo4 PFU/ml. About 60% of the presumptive p mutants (those giving a double negative on PK-2a minilayers) were found on passage to contain no virus. Of the remaining 40%, nearly all proved still to be p mutants after their passagein CEF at 34.5”, and most of the isolates originally classified as ts mutants still showed mutants phenotype after their low temperature pass. ts mutant’s were isolated at a frequency of 2.3 % and p mutants 1.8%. On retesting, many of the ts but not the p mutants showed relatively high plating efficiencies under the restrictive conditions (> 10M2of the plaque count under permissive conditions), probably becauseof leak and/or reversion. These mutants were rejected as unsuitable for further genetic analysis. Stocks of suitable p and ts mutants were numbered consecutively and stored at -70”. They were stable during further passages in CEF at 34.5’, and those ts and p mutants tested were just

596 as sensitive to inhibition parental wild-type virus.

SAMBROOK,

by BDU

PADGETT,

as the

The Frequency of ts Mutants in Virus Grown in the Presence of BDU Although usable ts mutants were not common in the mutagen-treated virus stocks (about 1% of all plaques picked) there were indications Ohat a much higher proportion than this of the BDU-treated virus were in fact ts mutants. A detailed examination was made of the yield of virus after passage at 34.5” in the presence of 1.5 pg BDU per millilitre. Two hundred plaques were picked from the yield and were compared with 200 wild-type plaques from a control preparation passed in the absence of BDU. Each isolate was inoculated onto PK-2a minilayers in two separate t’rays at two dilutions, usually undiluted and 1 in 5 or 1 in 10. Duplicate sets of minilayers were incubated at 34.5” and 39.5”. For each isolate the total number of infective particles picked, and also the ratio of plaques formed at 39.5 to 34.5”, was determined. The results are shown in Figs. 2 and 3.

AND TOMKINS

It is seen that the number of infective particles recovered from the wild-type plaques varied between 20 and 450, the mode being 239. The number recovered from the BDU-passed virus plaques on the other hand varied between 0 and 190 per plaque with a mode of 41. it seems therefore that most of the viable particles formed in the presence of BDU have a reduced capacity for multiplication (at least in PK-2a cells). These same isolates, in addition, are more heat defective than those obtained from the wild-type control in that their efficiency of plating 39.5”: 34.5” is distributed around a mean of 0.4 whereas the wild-type isolates are distributed around a mean of 1.0. The conclusion reached from this experiment is that the yield of virus produced in the presence of BDU consists mainly of mutated virus particles. Isolation of ta Mutants sure against RP+

with Selection Pres-

The genome of rabbitpox virus may consist of as many as 200 cistrons and, therefore, a large number of different ts and/or

rl200

300

400

I 500

PFU per ISOLATE

2. A comparison of the number of infective particles per plaque isolate from untreated RP+ and from RP+ grown in the presence of 5-bromodeoxyuridine. Two hundred plaques were picked from RP+ grown in the presence of 1.5 pg BDU per millilitre and compared with 200 wild-type plaques picked from a control preparation passed in the absence of BDU. Each isolate was inoculated onto PK-2a minilayers, and the plaques were counted. The histogram shows the percentage of isolates containing different numbers of infective particles. FIG.

507

ISOLATION OF MUTANTS OF RABBITPOX VIRUS

RP+

’

+-b

, .--, 0.2

0.4

0.6

0.8

PLAQUE

COUNT COUNT

i-l 1.0 39.5O 34.F

1.2

1.4

FIG. 3. The efficiency of plating at 34.5” and 39.5” of plaque isolates from untreated RP+ and from RPf grown in the presenceof 5-bromodeoxyuridine. The same400isolates described in Fig. 2 were inoculated onto each of two PK.2a minilayers and incubated at 34.5” or 39.5”. After 65 hours the minilayers were stained and the ratio of plaques at 39.5” to plaques at 34.5” was determined for each isolate. The histogram shows the percentage of the isolates with different efficiencies of plating at the two temperatures.

p mutants would be required for mapping the genome. The labor involved in collecting enough mutants would be reduced if the proportion of mutants in those plaques picked for testing were increased. This could be accomplished by increasing the number of mutants in the population by growing the virus stock in higher concentrations of mutagen, but such a procedure increases the risk that the mutants isolated will be “doubles” and therefore of little use in genetic analyses. The desired result could also be obtained without altering the absolute number of mutants in the population by applying selection pressure in favor of the mutants at the time of isolation; that is, by preventing or inhibiting the growth of nonmutant clones. A method, based on the following rationale, was devised for applying selection pressure in favor of ts mutants. We assume that at 39.5” ts mutants undergo a normal sequence of replicative events until the step controlled by their mutated gene is reached. At this point their replication stops, but they may be rescued if the temperature is lowered to 34.5” within a reasonable period of time. Certain inhibitors of poxvirus replication are available which (a) have no

permanent effect on the ability of uninfected cells to support virus growth subsequently and (b) whose effect on viral replication is the formation of noninfectious virus particles so that their action is irreversible. Of these inhibitors, isatin fl-thiosemicarbazone (IBT) was chosen because it affects a late stage in the replication of poxviruses (Appleyard et al., 1965) by preventing normal translation of “late” viral messenger RNA (Woodson and Joklik, 1965). Preliminary experiments with RP+ and some previously isolated ts mutants led to the following procedure, which is a compromise between those conditions under which IBT was most effective in preventing growth of RP+ and those permitting maximum survival of ts mutants. To increase the synchrony of infection, virus was allowed to adsorb onto PK-2a cells for 2 hours at 4“ followed by 30 minutes at 37”. The cell sheets were washed once, and maintenance medium containing 60 pM IBT was added. After 16 hours’ incubation at 39.5’ the medium was removed, the cell sheets were washed twice with 15 minutes’ incubation at 37” between washes, agar overlay was added, and incubation continued at 34.5” for 3 days before neutral red was added.

598

SAMBROOK,

PADGETT,

The number of ts mutants in 1084 plaques that developed after treatment with IBT was compared with that from 1088 randomly picked plaques from control plates incubated without IBT. Twenty-six ts mutants were isolated using IBT and 6 without IBT, so that use of IBT resulted in a fourfold increase in the proportion of ts mutants among the plaques picked. DISCUSSION

The multiplication of rabbitpox virus in the presence of BDU and AP results in a yield of virus decreased in infectivity but. increased in its proportion of conditional lethal mutants. The results with BDU indicated that most of the yield of virus grown in the presence of the mutagen had been mutated, but only a small proportion of the mutated virus showed the ts character with sufficient penetrance to be scored as such. There ic little intrinsic interest’ in the formal exercise of collecting mutants per se; the objective in this work is to use them as tools for genetic analysis. To do this the mutants should satisfy three further conditions. 1. They should not be “sister” mut’ants; that is, they should not suffer from an identical genetic defect. The collection of sister mutants is likely to occur only by selection from preexisting mutant clones in the original wild-type stock. In bhe present case the original cloning was carried out under conditions that selected against mutants of the type sought’, and the experimental evidence suggests that the mutagens have in fact induced the mutants. 2. The mutants should not be LLdoubles,” that is, they should each carry only one mutated site demonstrable by the particular selective conditions imposed, for with double mutants both genetical and physiological mapping are difficult. BDU and AP are believed to act as mutagens by inducing A-T transitions at individual base pairs in the DNA (Freese, 1963). Since single transitions of this sort are known to cause conditional lethal mutations in T4 of both ts and am phenotypes (Epstein et al., 1963), there seems no reason why they should not do so n rabbitpox virus. Preliminary results with

AND

TOMKINS

our ts mutants indicates that they are not double mutants. 3. To map rabbitpox virus using conditional lethal mutants it is necessary that these mutat’ions can occur at many sites within the virus genome. With the ts mutants, preliminary results indicate that this condition is fulfilled, but the succeeding paper (Fenner and Sambrook, 1966) suggests that the p mutants are not the equivalent of amber mutants and may be clustered in one cistron. The use of inhibitors acting earlier than IBT in the growth cycle of the virus may facilitate the isolation of particular classes of ts mutants. REFERENCES APPLEYARD, G., HUME, V. B. M., and WESTWOOD, J. C. N. (1965). The effect of thiosemicarbazones on the growth of rabbitpox virus in tissue culture. Ann. Acad. Sci. N.Y. 130, 92-104. BURGE, B. W., and PFEFFEREORN, E. R. (1965). Condit,ional lethal mutants of an animal virus: identificat’ion of two cistrons. Science 148, 959960. COOPEX, P. D. (1965). Rescue of one phenotype in mixed infections with heat-defective mutants of type 1 poliovirus. virology 25, 431-438. EASTERBROOK, K. B. (1962). Interference with maturation of vaccinia virus by isatin @-thiosemicarbazone. Virology 17, 245-251. EASTEKBROOK, K. B., and D.4VERN, C. I. (1963). The effect of 5-bromodeoxyuridine on the multiplication of vaccinia virus. virology 19, 509-521. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M., I~ENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375392. FENNEIL, F. (1958). The biological characters of several strains of vaccinia, cowpox and rabbitpox viruses. Virology 5, 502-529. FENNER, F., and SAMBROOK, J. F. (1964). The genetics of animal viruses. Ann. Rev. Microbial. 18, 47-94. FENNER, F., and SAMBROOK, J. F. (1966). Conditional lethal mutants of rabbitpox virus. II. Mutants (p) that fail to multiply in PK-2a Cells. Virology 28, 600-609. FHEESE, E. (1963). Molecular mechanism of mutation. In Molecular Genetics (J. H. Taylor, ed.), Part 1. Academic Press, New York. INOUE, Y. K., and KATO, H. (1963). Studies on

ISOLATION

OF MUTANTS

Japanese B encephalitis virus. V. A thermoefficient mutant of Japanese B encephalitis virus. Virology 21, 222-225. MCCLAIN, M. E. (1965). The host-range and plaque morphology of rabbitpox virus (RPu+) and its u mutants in chick fibroblast, PK-2a and L929 cells. Australian J. Exptl. Biol. Med. Sci. 43, 3144.

OF RABBITPOX

VIRUS

599

M. E., and GREENLAND, R. M. (1965). Recombination between rabbitpox virus mutants in permissive and nonpermissive cells. Virology 25, 516-522. WOODSON, B., and JOKLIK, W. K. (1965). The inhibition of vaccinia virus multiplication by isatin B-thiosemicarbazone. Proc. Natl. Acad. Sci. U.S. 54, 946-953. MCCLAIN,