Isolation and partial characterization of temperature-sensitive mutants of Mengo virus

VIROLOGY

70, 190-194

Isolation

(1976)

and Partial

Characterization of Temperature-Sensitive Mutants of Mengo Virus

DONALD N. DOWNER, SYLVIA SUNDERLAND, Department

of Biochemistry,

Uniuersity

AND

JOHN S. COLTER

of Alberta, Edmonton, Alberta,

Canada

T6G 2H7

Accepted October 3, 1975

A total of 18 temperature-sensitive mutants of Mengo virus, produced by chemical mutagenesis, has been partially purified and characterized with respect to (1) single cycle growth at the permissive (33”) and nonpermissive (39”) temperatures, (2) ability to stimulate the synthesis of viral ribonucleates at the two temperatures, and (3) thermal stability. Of the 18, 11 and 7 were found to be RNA- and RNA+, respectively, and 5 were found to have significantly lower thermal stabilities than the wild-type.

Current genetic studies with animal viruses are based in large measure on the use of temperature-sensitive (ts) mutants, and conditional lethal mutants of this class have been used for genetic and biochemical investigations of representative members of a number of groups of mammalian viruses (l-19). In this communication we describe the production, isolation, and preliminary characterization of a number of ts mutants of Mengo virus. A similar report has been published recently by Bond and Swim (20). Cells of Earl’s L-929 strain of mouse fibroblasts were grown in suspension culture in Ca2+-free Eagle’s minimum essential medium (MEM) and as monolayers in Eagle’s basal minimum essential medium (BME), both media supplemented with 5% horse serum. Monolayer cultures, for plaque assay and the determination of growth curves, were grown in either 10 x 35mm or 15 x 60-mm plastic petri dishes (Falcon), and for the production of large pools, in 110 x 425-mm roller bottles. The wild-type (wt) virus employed was the plaque variant of Mengo virus designated M-Mengo (211. It was propagated in L cells and purified by the method described in Ziola and Scraba (22). Chemical mutagenesis and the selection of ts mutants were carried out by proce-

dures essentially the same as those described by Fields and Joklik (5). Purified virus (about 10’” PFU/ml) was incubated for 1 hr at 25” in 0.1 M Na phosphate, pH 7.6, containing 0.018% N-methyl-N’-nitroN-nitroso-guanidine (NTG) after which the suspension was diluted with an equal volume of Na-phosphate buffer containing 5% glycerol and dialyzed overnight against SSC. Nitrous acid (NA) mutagenesis was achieved by incubating the virus for 35 min at 20” in 1.0 M acetate buffer, pH 4.4, containing 0.67 M sodium nitrite, and then diluting the suspension loo-fold with Tris-acetate buffer, pH 8.1. Confluent monolayers of L cells were infected with 10-20 PFU of the mutagenized preparations and incubated for 72 hr at 33” (permissive temperature), at which time (after staining the monolayers with neutral red) the circumference of each well-isolated plaque was marked and incubation was continued at 39” (nonpermissive temperature). After 24 hr at 39” each plaque that had not increased in size was picked into 1 ml of PBS and the suspension was frozen and thawed repeatedly to free virus from the agar plug. Each suspension was assayed for infectious virus at 33 and 39, and those isolates that exhibited a 331 39” plaque ratio of 1O:l or greater were retained for further study.

190 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

191

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tures, and with respect to thermal stability. To determine growth curves and examine viral-RNA synthesis, replicate monolayer cultures (in 10 x 35mm dishes) were infected at a m.o.i. of 10 PFU/cell and then incubated (at 33 or 39”) in 1 ml of BME containing 2 &i 13Hluridine and 3 kg actinomycin D. Cultures were harvested at intervals and the cells disrupted by sonic vibration. Virus production was determined by plaque assay at 33”, and viralRNA synthesis was monitored by measuring TCA-precipitable radioactivity in aliquots of the cell lysates. In each experiment, the behavior of one or two ts mutants was compared with that of wt virus, and RNA synthesis was monitored in mock-infected cultures at both temperatures. Those mutants with which incorporation of uridine at 39” did not exceed the level observed in uninfected cells were designated RNA- mutants. With RNA+ mutants, uridine incorporation at 39” did not differ significantly from that observed with wt virus. Data obtained with ts 100, an RNA+ mutant, are shown in Fig. 1. At 33”, both virus production and uridine incorporation

Suspected ts mutants were passaged three times in monolayer cultures at 33” (using virus from a single plaque at each passage) before plaques were picked and used to infect monolayers in plastic tissue culture flasks (Falcon, growth area = 75 cm2; 10 plaques/flask). If virus from these cultures was found to have retained its ts character, .it was used as inoculum to produce larger stocks by infecting roller bottle cultures. Virus from roller bottle cultures was concentrated and partially purified by a method that involved precipitation of the virus from clarified lysates with methanol (final concentration = 20%), incubation of the methanol precipitate for 30 min at 20 with chymotrypsin (80 wglml), clarification of the suspension by extraction with CHCl,, and sedimentation of the virus through a discontinuous sucrose gradient. Of several thousand plaques produced by suspected ts mutants, 290 exhibited a 33139”plaque ratio of 10 or greater, and of these 56 passed the more rigorous screening procedure. Of this group, 18 have been purified from roller bottle pools and characterized with respect to growth and ability to stimulate viral-RNA synthesis at the permissive and nonpermissive tempera1s IOO(RNA+) r

33°C

1s

at c

4>o” 0

2

4

6

8

ts

IO 12 0 2 HOURS POSTINFECTION

4

6

8

IO

12

FIG. 1. Growth curves of wild-type and an RNA+ mutant of Mengo virus. Infected monolayers were incubated at 33 or 39” in medium containing 13Hluridine. Samples were removed at intervals, the cells lysed by sonic vibration and assayed for infectious virus and TCA-insoluble cpm. Triangles: virus; circles: ts mutant. Closed symbols: infectious virus; open symbols: TCA-insoluble cpm.

wt

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9il

L” 20

33°C

1s 506U?NA-1 506 (RNA-)

,’ ,’

39°C

1s 506 (RNA-1 (RNA-)

;i == z

66-

- 16% n\ ‘9 x

??-

- 12 ”: - I2 ” z 0 t2

? P 0 5

6-

-02 8 ii !i

5-

-4g 2 ,I

0

2

4

6

6

IO I2 12 0 2 HOURS POSTINFECTION

FIG. 2. Growth curves of wild-type and an RNA- ts incubated at 33 or 39” in medium containing [SHluridine. by sonic vibration and assayed for infectious virus and mutant. Closed symbols: infectious virus; open symbols:

in wt- and mutant virus-infected cells were comparable, whereas at 39”, although uridine incorporation was essentially the same in mutant- and wt virus-infected cells, there was virtually no production of mutant virus. It might be noted that, in all such experiments, uridine incorporation in cells infected with wt virus was found to be lower at 39” than at 33”, although virus production at the two temperatures was essentially the same. Comparable data obtained with the RNA- mutant, ts 506, are illustrated in Fig. 2. At 33” both virus production and uridine incorporation in cells infected with the mutant and wt viruses were very similar, while at 39” neither virus production nor uridine incorporation was detectable in cells infected with the ts mutant. Data obtained from studies of all 18 ts mutants are summarized in Table 1. Of the 18 (14 resulting from NA and 4 from NTG mutagenesis) 11 were found to be RNA- and 7 to be RNA+. The data in column 6 were obtained by titrating the partially purified roller bottle pools at the two temperatures and expressing the results as ratios. They range from a high of 7 x 10m2 forts 506 to a low of about 10h7forts 106, with the majority being in the 10e3to

4

6

6

IO

0

I2 12

mutant of Mengo virus. Infected monolayers were Samples were removed at intervals, the cells lysed TCA-insoluble cpm. Triangles: wt virus; circles: ts TCA-insoluble cpm. TABLE

1

PROPERTIES OF TS MUTANTS OF MENCO VIRUS -__ T Virus Percent. Plaquing efiViralstrain 3ge of 1~1 NA syn ciency Yield hesis at (39133”) 39” 39" wt +ve 8 x 10-l 106 26 .03 6.6 x 10-8 -ve 135 6.0 x 1O-5 38 .20 -ve 423 8.0 x 10-e 36 .lO -ve 506 78 .Ol 7.0 x 10-Z -ve 520 200 .50 1.0 x 10-Z -ve 525 1 .06 -ve 4.0 x 10-4 574 3 .60 -ve 5.0 x 10-2 620 N.A. 30 .05 2.0 x 10-S -ve 625 N.A. 3 .30 -ve 1.0 x 10-4 677 N.A. 29 .30 -ve 1.3 x 10-Z 1100 115 .40 -ve 3.0 x 10-S 430 26 .40 7.0 x 10-Z +ve 641 50 .12 4.0 x 10-a +ve 648 +ve 3.0 x 10-a 5 .04 676 1.0 x 10-s 9 .Ol +ve 100 fve 5.0 x 10-Z 15 .02 200 1.0 x 10-G 50 .Ol fve 500 13 .lO +ve 8.0 x 1O-4

1O-5 range. The data in columns 3 and 4 were obtained from single cycle growth experiments, such as those illustrated in

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Figs. 1 and 2, and show the yield of each ts mutant at 33 and 39” expressed as a percentage of the yield of wt virus obtained at the same temperature in the same experiment. Even at the permissive temperature, most ts mutants were found to replicate less efficiently than does the wt virus, the one notable exception being ts 520. The thermal stability of our ts mutants was examined by incubating virus samples (initial concentration = lo7 PFU/ml) in BME at 33, 39, and 50”, removing samples at intervals, and estimating surviving virus by plaque assay at 33”. The results are summarized in Table 2. Of the 18 ts mutants, 5 (ts 135, 525, 641, 676, and 200) appear to have a significantly lower thermal stability than the wt virus, suggesting that they have ts defects in one or more of their structural polypeptides. Two of the 5 are RNA- mutants, which suggests that in addition to an altered structural polypeptide they may have a mutation in that segment of the genome which codes for the nonstructural polypeptide required for the production of the viral-RNA replicase. The data presented here do not provide any information regarding either the preTABLE THERMAL

STABILITY __---~-

2 OF MENCO

Half life in minutes

135

120

423 506 520 525 574 620 625 677

480 570 300

600 540 70 360 435 180 70 330 390 420 300 220 540 70 390 80 390 55 300

430 641 648 676 100

200 500

1.

R. W., and 41-49 (1968).

SIMPSON,

HIRST,

G. K.,

Virology 35,

2. MACKENZIE, J. S., J. Gen. Viral. 6, 63-75 (1970). 3. COOPER, P. D., Virology 35, 584-596 (1968). 4. COOPER, P. D., STANCEK, D., and SUMMERS, D. F., Virology 40, 971-977 (1970). 5. FIELDS, B. N., and JOKLIK, W. K., Virology 37, 335342 (1969). 6. ITO, Y., and JOKLIK, W. K., Virology 50, 189207 7. PRINGLE, C. R., and DUNCAN, I. B., J. Viral. 8, 5661 (1971). 8. WUNNER, W. H., and PRINGLE, C. R., Virology

12. WILLIAMS,

720 600

30

106

1100

REFERENCES

18

At 50”

100

We wish to thank Carol Campbell, Pat Carpenter, Irene Korolak, and Mr. Conn Lee for valuable technical assistance, and Mr. Perry D’Obrenan and Cathy Hicks for preparing the figures shown herein. The studies were supported by Grant Number MT-1191 from the Medical Research Council of Canada. S. S. was the recipient of an MRC Studentship.

8 40

At 39”

400 420 480 540 660 600 100 600 95 420 60 330

ACKNOWLEDGMENTS

48, lOCll1 (1972). B. W., and PFEFFERKORN, E. R., Virology 30, 204-213 (1966). 10. BURGE, B. W., and PFEFFERKORN, E. R., J. Mol. Biol. 35, 193-205 (1968). 11. TAN, K. B., SAMBROOK, J. F., and BELLETT, A. J. D., Virology 38, 427-439 (1969).

At 33

wt

cise biochemical lesion associated with any of the ts mutants or the biological function of any of the four or five virus-specific, nonstructural polypeptides synthesized in Mengo-infected cells (23). Such information may come from careful analyses of the synthesis of viral macromolecules at 33 and 39” and during temperature shifts. Studies of this nature are in progress.

(1972).

OF TS MUTANTS VIRUS ~__-

Virus strain

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15

28 8 40 15 13 15 20 25 13 24

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J. F., GHARPURE, M., USTACELEBI, MCDONALD, S., J. Gen. Viral. 11, 95101 (1971). ESPARZA, J., PURIFOY, D. J. M., SCHAFFER, P. M., Virology 57, A., and BENYESH-MELINCK,

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MACKENZIE, J. S., SLADE, W. R., LAKE, J., PRISTON, R. A. J., BISBY, J., LAING, S., and NEWMAN, J., J. Gen. Virol. 27, 61-70 (1975). 15. EKHART, W., Virology 38, 120-125 (1969). 16. DIMAYORCA, G., CALLENDER, J., MARIN, G., and GIORDANA, R., Virology 38, 126133 (1969). 17. TEGTMEYER, P., and OZER, H. L., J. Viral. 8,

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18.

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KAWAI, S., and HANAFUSA, H., Virology 46, 47b479 (1971). 20. BOND, C. W., and SWIM, H. E., J. Viral. 15, 286 296 (1975). 21. ELLEM, K. A. O., and COLTER, J. S., Virology 15, 19.

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(1961). R., and SCRABA, D. G., Virology 57, (1974). E., SEEHAFER, J., and COLTER, J. S., Virology 61, 315-326 (1974).