Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants

VIROLOGY

119,372-381 (1982)

Complementation and Genetic Linkage between Virus Temperature-Sensitive Mutants R. DRILLIEN,’

D. SPEHNER,

Vaccinia

AND A. KIRN

Groupe de Rechm-ches de UNSERM (U 74) sur la Pathog&ie o!esIqfections Virales et Laborato-ire a!~ Virolqrie de la Facult6 de M&&wine, Univmiti Louis Pasteur, S, rue Koeberlk, Stqw.sbourg, France Received November 23, 1981; accepted February 26, 1982 Vaccinia virus temperature-sensitive (ts) mutants were isolated after nitrosoguanidine mutagenesis. A number of these mutants exhibited host range temperature sensitivity in that the efficiency of plaque formation at the nonpermissive temperature was poorer on chick cells than on hamster or human cells. Forty-two mutants were assigned to 23 different complementation groups on the basis of complementation and the efficiency of apparent recombination at the nonpermissive temperature. Recombination frequencies were also determined from mixed infections carried out at the permissive temperature and it was confirmed that mutants within the same complementation group recombined less efficiently with each other than mutants belonging to different groups. Mutants from two of the largest groups could be tentatively ordered on linear intragenic maps that spanned 0.8 and 2 recombination units. Moreover, from intergenic crosses between mutants in 14 different complementation groups, a linkage map spanning 66.3 recombination units, was derived. This study illustrates the feasibility of two-factor recombination mapping of poxvirus mutations and provides genetic data that should be of relevance in further analysis of the ts mutations.

studies with rabbit pox ts mutants demonstrated the occurrence of efficient recombination but the results did not allow map construction (Padgett and Tomkins, 1968). Since these reports additional vaccinia virus and rabbit pox ts mutants have been isolated (Dales et al., 1978; Chernos et al., 1978; Ensinger, communication from 2nd Poxvirus-Iridovirus Workshop, 1978; Lake and Cooper, 1980; Condit and Motyczka, 1981). In several cases mutants have been assigned to different complementation groups (Ensinger, communication from 2nd Poxvirus-Iridovirus Workshop, 1978; Lake et al., 1979; McFadden and Dales, 1980; Condit and Motyczka, 1981). As yet however, few studies have been concerned with the possibility of recombination mapping of poxvirus mutants. Chernos et al. (1978) have presented data from which a genetic map comprising seven mutants was established and Lake et al. (1979) have mapped relative to one another a series of five ts mutants that display a similar morphogenetic phenotype. In order to under-

INTRODUCTION

In early studies with poxviruses, attempts were made to order mutations on genetic maps according to recombination frequencies. The white pock u mutants of rabbit pox could be arranged into linkage groups on the basis of positive or negative recombination, the exact frequency of recombination being extremely variable with the test system employed (Gemmell and Cairns, 1959; Gemmell and Fenner, 1960). More recent data have shown that the white pock mutants of rabbit pox result from deletions and transpositions of viral DNA suggesting that mutants which had failed to recombine were deleted or interrupted in overlapping sequences (Moyer and Rothe, 1980; Moyer et al., 1980). Temperature-sensitive (ts) mutations can without any doubt affect a much wider range of essential viral functions than white pock mutations. The first genetic ’ To whom reprint

requests should be addressed.

0042-6822/82/080372-10$02.00/O Copyright Q 1999 by Academic All rights of reproductionin

Press. Inc. reserved.

anyform

372

VACCINIA

VIRUS TEMPERATURE-SENSITIVE

100

h x V

10

ti E 1 3 a >

0.1

0.01

,.

1

10

20 NG

I

1.

30

40

5c

(m/ml

)

FIG. 1. NG inhibition of vaccinia multiplication. Cell cultures were infected and incubated in the presence of NG at various doses as described under Methods. After 30 hr of infection the yields were titrated at 33”. The virus titers in the mutagen-treated cultures were calculated as percentages of the titer in the control infection.

take a detailed genetic study of vaccinia virus we have recently isolated a set of ts mutants after nitrosoguanidine mutagenesis. The temperature sensitivity of the mutants has been determined on several host cell systems and the mutants have been assigned to different complementation groups. Two-factor intragenic and intergenic crosses were carried out at the permissive temperature and in a number of cases the recombination data have allowed the construction of linear linkage maps. MATERIALS

AND METHODS

Cells. Primary chick embryo fibroblasts (CEF), baby hamster kidney cells (BHK 21), and human embryonic fibroblasts, line 809 (HEF), were used in this study. CEF and HEF were grown in Eagle’s basal medium (Eurobio) supplemented with 10%

MUTANTS

373

calf serum and bactotryptose phosphate broth. BHK 21 cells were grown in BHK medium (Eurobib) supplemented with 10% calf serum. Virus. The vaccinia virus Copenhagen strain was plaque purified prior to mutant isolation as previously reported (Drillien et al., 1981). Plaque assays were carried out at 33” (permissive temperature) or 39.5” (nonpermissive temperature) for 48 hr under liquid medium supplemented with 5% calf serum except when mutant isolation or virus cloning were undertaken, in which case solid medium containing 1% Noble agar and 5% serum was used. Mutagenesis and mutant isolation. Confluent CEF monolayers were infected with approximately 0.5 PFU per cell. After one hour of virus adsorption at room temperature the inoculum was removed and cells were covered with fresh medium to which various concentrations of N-methylN’-nitro-N-nitrosoguanidine (NG) were added. Incubation of infected cells was then at 33” for 30 hr. Temperature-sensitive mutants were isolated from the mutagenized stocks by a plaque enlargement technique. Briefly, plaques were formed on CEF under a 1% agar overlayer. Two days after infection at 33” the cells were stained with 0.005% neutral red in medium containing 1% agar. Three days after infection the plaques could be visualized and they were outlined with a felt-tipped pen. TABLE ISOLATION

NG (ehnl) 0 10 20 25

OF ts MUTANTS

Virus plaques” 563 324 230 198

Plaques testedb 1 24 24 28

1

AFTER NG

Mutants isolated’ 0 10 11 14

MUTAGENESIS Isolation frequency (%I <0.2 3.1 4.8 7.1

“This figure represents the total number of plaques obtained on the dishes. *This figure represents the number of virual plaques that failed to enlarge after the shift to the high temperature. ‘Some of the mutants have not been included in this table since they were taken from dishes whose plaque number had not been counted.

374

DRILLIEN,

SPEHNER,

The infected dishes were then shifted to 39.5” for 2 days and the plaques that failed to enlarge were picked. Potential ts plaques were individually grown to higher titers and tested in a plaque assay for temperature sensitivity. All mutants retained were cloned once again on CEF under an agar overlayer. Mutant numbers were preceeded by the letter N to denote that they were isolated after NG mutagenesis. Comphrwntation test. Dishes containing 24 wells (2 cm2) were seeded with approximately 300,000 secondary CEF per well. After incubation at 37” for one day the medium was removed and cells were infected with 0.1 ml of a single mutant or 0.1 ml of two different mutants. Each virus was added at a multiplicity of about 5 PFU per cell. After virus adsorption at 4“ for one hour the inoculum was removed and

OF INFECTIVITY

2 (39.5’/33”)

Mutant

CEF

BHK

HEF

Mutant

Wild type ts Nl N2 N3 N4 N5 N6 N7 Nl3 NS NlO Nil N12 N13 N14 N15 N16 N17 N18 N20 N21 N22

7.10-i 3.1o-5 2.10-5 2.10-’ 9.10-a 2.10-5 9.1o-5 3.10+ 5.10-4 1.10-4 4.10-5 2.10-4 2.10-5 1.10-5 1.10-4 7.10-5 3.1o-6 2.1o-6 2.10-6 3.10+ 5.10+ 3.10+

9.10-i 2.10-s 3.10-* 6.10-3 1 3.10-l 5.10-2 1.1o-s 3.10-l 1.10-4 3.10-l 7.10-5 3.10-* 3.10-4 1.10-l 5.10-4 9.10-2 5.10-s 2.10-5 3.10-2 9.10-s 1.10-l

310-l 4.10-5 4.10-4 4.10-4 9.10-2 2.10-2 9.10-2 1.10-’ -

ts N23 N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N36 N37 N38 N39 N40 N41 N42 N46 N47

2.10-l 4.10-2 3.10-s 1 7.10-3 6.10-5 5.10-4 3.10-a -

KIRN

cells were rinsed twice with one ml of PBS. Medium was aded to each well and the cultures were incubated at 39.5’ for 24 hr then frozen. Virus in each well was titrated on CEF at 33” and 39.5” after thawing cells and disrupting them for 15 set with an MSE ultrasonic drill. A complementation index (C.I.) was calculated using the formula (A X B)33/((A)33 + (B)33) in which (A X B)33 is the yield of the mixed infection titrated at 33” and (A)33 + (B)% is the sum of the yields of the corresponding single infections also titrated at 33”. The above formula was chosen instead of the related one ((A X B)% - (A X B)3s.5) X ((A)% + (B)%) which eliminated from the end result the contribution of wildtype-like plaques that arise in the mixed infection. Since it was found that the value of (A X B)3s.5 was low in crosses involving

TABLE RATIO

AND

CEF

BHK

HEF

1.10-’ 2.10-4 3.1o-6 2.1o-6 3.1o-5 3.10-5 1.10+ 3.1o-5 1.10-4 9.10-6 7.10-S 4.10-3 1.1o-5 4.10-4 6.10-5 2.10-” 2.10-5 6.10-’ 3.1o-6 9.1o-6 9.1o-5

2.10-2 2.10-I 6.10-5 6.10-4 3.10-3 5.10-3 1 1.10-’ 5.10-4 2.10-4 2.10-4 2.10-3 5.10-3 2.10-l 1.10-4 6.10-4 1.10+ 3.10-3 3.10-3 1.10-’ 1.10-*

6.10-3 5.10-2 5.10-4 2.10-l 2.10-4 5.10-” 8.10-4 9.10-* -

a Virus stocks were titrated at 39.5“ and 33’ and the ratio of the number of PFU at39.5O over the number at 33” was calculated. In some cases where cell necrosis obscured plaque counts a high m.o.i., the values given are the highest possible ratios. The titers of virus stocks at the permissive temperature varied from one mutant to another over the range of 1.106 to 1.10’ PFU/ml. None of the mutants displayed a host range phenotype at the permissive temperature.

VACCINIA

VIRUS

TEMPERATURE-SENSITIVE

TABLE

MUTANTS

375

3

COMPLEMENTATION~ AND RECOMBINATION~ IN CROSSESAT 39.5” Complementation

Y

group

(0.2)

25

(0.12)

(0.24)

1.0 (0.10)

(1.02)

27

10

13

18 25.3 (30.71

13 18

0.8

1E5) Complementation

ts mutant N

7

1.6 (0.181

22 3.2 (26)

0.8 (0.51)

group

26

28

15.0 (25.9)

(17.91

3.0

1.4

1.4

10.481

10.241

1.1

0.9

(0.441

(0.18

(1.2)

1.1 CO.08

(0.261

26 28

1.8

11

42 12.2 (27.31

1.1 10.88)

1.2 1.5 CO.581

(:::, 1.5 (3.05)

‘sbComplementation indexes (in boldface) and the percentage of wild-type recombinants in the yield (in parentheses).

mutants in the same complementation group and much higher in crosses between mutants in different groups, excluding it from the calculations tended to enhance the distinction between complementing and noncomplementing mutants. The frequency of appearance of presumptive wild-type recombinants in the complementation test was calculated as the ratio of the yield of the mixed infection titrated at 39.5’ over the yield of the mixed infection titrated at 33”. In the case of single infections this ratio was so low that

it did not significantly contribute to the values obtained from mixed infections. Recombinatim frequencies. The frequency of recombination at the permissive temperature was determined from mixed infections in experiments designed similarly to the complementation test except that adsorption was at room temperature, incubation at 33”, and ultrasonic treatment was for one minute followed by immediate dilution of virus to avoid clumping. Recombination frequencies were calculated with the formula ((A X B)59.5/(A

376

DRILLIEN,

SPEHNER,

B)%) X 2 X 100% where (A X B) is the yield of the mixed infection titrated at 39.5” or 33”. Similar calculations for control single infections gave values too low to contribute significantly to the results from mixed infections. The recombination frequencies were multiplied by two in order to account for non-wild-type recombinants that presumably occur in the same amount as wild-type recombinants but remain undetected. To enable a better comparison of results from different experiments recombination frequencies were also corrected for the twofold experimental variation of the ratio of the titer of the wild-type determined at 39.5’ over its titer at 33”. Multiplicities (5 PFU/cell) and the duration of infection (24 hr) were found to be optimal for recombination. At least four independent experiments were performed to determine frequencies. Standard deviations from the mean values were calculated according to the formula [(C Rp)/N - (C Rf)2/N2]1’2 where Rf is the recombination frequency and N the number of independent determinations.

AND

KIRN TABLE

X

COMPLEMENTATION Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

4 GROUPS Mutants

1

2, 8 3 4 5, 31 6, 36 7, 9, 25, 27, 38 10 11, 14, 15 12, 30, 47 13, 18, 22, 26, 28, 33, 42 16, 17 20 21 23, 24, 36 29 32, 33 34 37 39 40 41 46

RESULTS

Mutant Isolation and Phenotypic Characterization All mutants used in this study were isolated after NG mutagenesis of infected cells. The mutagen inhibited virus multiplication in a dose-dependent manner (Fig. 1). At 25 rg/ml the yield obtained was equivalent to the input titer. At 50 pug/ml the mutagen probably inactivated input virus. The efficiency of mutant isolation increased as the concentration of mutagen increased and no mutant could be isolated from unmutagenized stocks (Table 1). In our experience nitrosoguanidine has been a much more potent mutagen than 5’-bromodeoxyuridine, nitrous acid, or uv irradiation although simultaneous experiments have not been conducted. The ratio of the titer of each mutant stock at 39.5“ over the titer at 33” was determined on CEF, BHK 21, and HEF (Table 2). All mutants had low efficiencies of plating on CEF at 39.5”. On BHK and on HEF some of the mutants displayed a

similar degree of temperature sensitivity as on CEF (e.g., ts Ni, N39) whereas the phenotype of others was less marked (e.g., ts Ns, N12) or even close to that of the wild type (e.g., ts NIO, Nd.

Establishment of Complementation Groups Mixed infections were carried out between mutants at the nonpermissive temperature and the complementation index (C.I.) and the frequency of appearance of wild-type plaques were calculated for each cross. Since more than 600 different crosses were performed only representative data are presented. Table 3 contains the results obtained for the two largest complementation groups identified. In each case mutant N 10 was arbitrarily chosen as a ts marker external to the complementation group being studied. Crosses between mutants expected from previous experiments of the same kind to be in the same complementation groups, gave in most cases C.1.s that were below 2. In crosses involv-

VACCINIA

VIRUS

TEMPERATURE-SENSITIVE TABLE

INTRAGENIC

5

RECOMBINATION’ AT 33” Complementation

I

ts

mutant

N

7

9

group

I

,

0.04 (0.031

25

0.60 CO. 44)

0.62 (0.441

0.46 (0.20)

0.42 (0.16)

27

0.42 (0.36)

Complementation

10

U

J*3 (3.21

I3

18

t

7

25

9

ts mutant N

377

MUTANTS

11

group

18

22

26

28

42

16.6 (5.0)

9 (2.41

12.5 (8.0)

8.0 (2.8)

12.3 (2.3)

0.68 (0.20)

1.04 CO. 541

0.82 10.30)

0.50 (0.191

1.04 (0.24)

0.82 (0.281

0.76 (0.321

0.88 (0.20)

1.96 (0.32)

’ Recombination frequencies are in bold face: standard the mean values are in parentheses.

ing mutants belonging to different groups (only those with mutant N10 in the results presented) C.1.s were higher than 2. The highest C.I. in these experiments was 25 but higher C.1.s were found in other crosses. In the intragenic mixed infections wild-type plaques appeared at a low frequency (less than 3% of the yields of the crosses). When the complementation index was low, but the percentage of wildtype plaques in the yield was high, for instance tsN9 X tsN10, mutants were allocated to different complementation groups

deviations from

on the basis that they were genetically too distant to belong to the same group. Subsequent recombination mapping at the permissive temperature has most often confirmed this assumption. Criteria such as those described above were used in the analysis of the mixed infections between 42 mutants which could thus be assigned to 23 different complementation groups (Table 4). At least two of the mutants (tsN33 and N36) may harbor two mutations since they failed to complement more than one group. The mutants were origi-

378

DRILLIEN,

SPEHNER, AND KIRN

nally isolated from eight independent infections at various doses of NG. Where sister isolates could be suspected the mutants were found to recombine with one another therefore excluding this possibility. Maps Obtained from Intragenic nation Frequencies

c~atbn

Group

S;J5-

Recombination Map of Mutants Belonging to D@went Complementation Groups Mutants belonging to different complementation groups were crossed by each other at the permissive temperature in a number of combinations and the recombination frequencies (Rf) were determined (Table 6). In most instances the Rfs were similar when two mutants in a single complementation group were crossed by another mutant outside this group as for mutants 7 and 25 or 16 and 17. Similarly to the intragenic crosses, standard deviations from the mean were quite large; however, the mutations could be ordered on a best fit linear linkage map on the basis of Rfs (Fig. 3). Additivity of map units was best for the closest markers. With increasing distances the Rfs reached a plateau level which was in the range of 20 to 30% recombination. Altogether the map which comprised mutants belonging

27

7

38

iila 0.60

Recombi-

To confirm the genetic proximity of mutations that belong to the same complementation group, mutants were crossed at the permissive temperature by each other and by an outside marker in all pairwise combinations. The intragenic recombination frequencies in these crosses were always much lower than the intergenic frequencies as illustrated for complementation groups 7 and 11 in Table 5. Although the standard deviations in these experiments were high most of the mutants could be tentatively ordered relative to one another on linear maps (Fig. 2). The maps of complementation groups 7 and 11 spanned, respectively, 0.8 and 2.04 recombinations units (obtained by summing the shortest intervals) and disnlayed significant albeit not perfect additivity.

7

030 0.42 LX?

Complementation

Group

11 42

28 -13

28$20.50 0.02

2.68 L 1.04 1.64 0.50 1.4 1.04

FIG. 2. Linkage maps of complementation groups 7 and 11. Mutants are represented by the boldface numbers. Horizontal lines separating mutant numbers are drawn to scale relative to one another using the data giving the shortest intervals. Recombination frequencies from the results in Table 5 are shown above a horizontal line separating the two mutants used in each cross.

to 14 different complementation groups spanned 66.3 recombination units. As for the nine other complementation groups, representative mutants have either failed to exhibit linkage to this map or the results are as yet insufficient. Mutants from groups 2 and 21 were linked to each other by about 7% Rf but they were unlinked to any other mutants. DISCUSSION

This study has enabled us to identify 23 complementation groups among 42 ts mutants of vaccinia virus. With regard to functional defects of the mutants only one (ts N41) was clearly unable to induce viral DNA synthesis at the nonpermissive temperature (not shown). Another DNA-neg-

VACCINIA

VIRUS

TEMPERATURE-SENSITIVE

TABLE INTERCENIC

7 -uz-WY_

RECOMBINATION~

6 5.0

“Recombination the mean values

8 10

379

6

Complementation

356

MUTANTS

AT 33” wow

12

13141617 ---w--w

202223

7 25 10 12 16 17 20 21 23 32 33 41 46 17.4

frequencies are are in parentheses.

ative mutant isolated after 5’-bromodeoxyuridine mutagenesis (ts Bll) has not yet been used in recombination studies; however, from complementation tests it comprises a distinct group from the previous ones. A number of the mutants isolated displayed host range temperature sensitivity in that they were more temperature sensitive on CEF than on BHK or HEF. Host range temperature sensitivity could be due to the existence within the permissive or semipermissive cells of protein activities equivalent to certain viral-induced activ-

114

in bold

face;

29 8.1

standard

6.6

deviations

from

ities. If this were true one would expect all mutants in a single complementation group to behave similarly. However, some complementation groups contained both host range ts mutants and non-host range ts mutants as for group 2 (mutants 2 and 8) and group 5 (mutants 5 and 31). These results suggest that host range temperature sensitivity may also be explained by the possibility of correcting a ts protein through interaction with cellular constituents. Such interactions would be favorable or not depending on the region of the viral protein that is mutated.

336

DRILLIEN,

SPEHNER, AND KIRN

In several instances where complementation was poor (a (3.1. below 2) mutants were assigned to different complementation groups in view of the fact that they recombined efficiently with each other at the nonpermissive temperature. This occurred for some crosses between mutants belonging to groups 8, 10, 14 and those belonging to groups 7 and 20 (results not shown). Since recombination mapping at the permissive temperature has suggested that mutants in the first three complementation groups are closely linked as well as mutants in the second two it is possible that these five groups make up only two different complementation groups. If this alternative is the correct one then these two complementation groups span quite large distances as judged by the recombination frequencies (6.6 between 8 and 14; 6.2 between 17 and 20). Further work, particularly biochemical studies of the ts defects may allow us to distinguish between these possibilities. In several other cases of poor complementation, the likelihood of mutants being in the same group could be dismissed since recombination showed that they were separated by other groups. Recombination mapping has proved to be a feasible task both in intragenic and intergenic crosses once a sufficient number of experiments were carried out. The major difficulty was clearly the variability in recombination frequencies. The fact that several of the complementation groups could not be linked to the intergenic recombination map is probably due to their localization in regions of the genome for which few mutants have been isolated. The significance and the usefulness of these maps will appear only upon further studies. As is not uncommon, they suffer from several inconsistencies. Whether the maps proposed are generally sound or not should eventually become clear through the use of the marker rescue technique as Sam and Dumbell (1981) have shown that this method may be applied to poxviruses. More recently the possibility of marker rescue mapping of poxvirus mutations has been demonstrated with cloned viral sequences (Condit and Motyczka, 1981b; Sam

20

x)122122

36

326

415

7

4617

6

ml

LL 6.6 b 15.9 150

-

35.0 ur L 17.6

3g 112 r

750 2l.6

2l.d IL0

-

20.6 2oh

-

35.2

58 Il.4

ja

-5.6 7.‘ 12.6

Y.0

12-2

12.d d9 8.1

10.6

2.4

32

5.0

112 5.6

3.0

7.7 7.0

r 58 2.9 3 16

FIG. 3. Linkage map of vaccinia ts mutants. Mutants are represented by the boldface numbers. Horizontal lines separating mutant numbers are drawn to scale relative to one another using the data giving the shortest intervals. Recombination frequencies from the results in Table 6 are shown above a horizontal line separating the two mutants used in each cross.

and Dumbell, personal communication). Furthermore, mapping may also be facilitated by the availability of ts mutations for which there is an associated alteration of the DNA restriction pattern as found for one vaccinia mutant (McFadden et al., 1980; Schtimperli et al., 1980). ACKNOWLEDGMENTS We are grateful to Francoise Koehren for help in the early phase of this study. This work was supported in part by a grant from the DRET, Contract 80440. REFERENCES CHERNOS,

V. I.,

BELANOV,

N. N. (1978). Temperature

E. F., and VASILIEVA, sensitive mutants of

VACCINIA

VIRUS

TEMPERATURE-SENSITIVE

vaccinia virus. I. Isolation and preliminary characterization. Acta fir01 22, 81-90. CONDIT, R. C., and MOTYCZKA, A. (1981a). Isolation and preliminary characterization of temperature sensitive mutants of vaccinia virus. Virology 113, 224-241. CONDIT, R. C., and MOTYCZKA, A. (1981b). “Fifth International Congress of Virology, Strasbourg” (Abstract). DALES, S., MILANOVITCH, V., POGO, B. G. T., WEINTRAUB, S. B., HUIMA, T., WILTON, S., and MCFADDEN, G. (1978). Biogenesis of vaccinia: Isolation of conditional lethal mutants and electron microscopic characterization of their phenotypically expressed defects. Virw 84, 403-428. DRILLIEN, R., KOEHREN, F., and KIRN, A. (1981). Host range deletion mutant of vaccinia virus defective in human cells. Virology 111, 488-499. GEMMELL, A., and CAIRNS, J. (959). Linkage in the genome of an animal virus. Virw 8.381-391. GEMMELL, A., and FENNER, F. (1960). Genetic studies with mammalian poxviruses. III White (u) mutants of rabbitpox virus. Virology 11, 219-235. LAKE, J. R., and COOPER, P. D. (1980). Synthesis of virus DNA and polypeptides by temperature sensitive mutants of rabbitpox virus. J. Gen. Viral 47, 243-259. LAKE, J. R., SILVER, M., and DALES, S. (1979). Biogenesis of vaccinia: Complementation and recombination analysis of one group of conditional lethal mutants defective in envelope self-assembly. Virology 96, 9-20.

MUTANTS

381

MCFADDEN, G., and DALES, S. (1980). Biogenesis of poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis. Virology 103, 68-79. MCFADDEN, G., ESSANI, K., and DALES, S. (1980). A new endonuclease restriction site which is at the locus of a temperature-sensitive mutation in vaccinia virus is associated with true and pseudoreversion. Virology 101, 277-280. MOYER, R. W., GRAVES, R. L., and ROTHE, C. T. (1980). The white pock (u) mutants of rabbit poxvirus. III Terminal DNA sequence duplication and transposition in rabbit poxvirus. Cell 22, 545-553. MOYER, R. W., and ROTHE, C. T. (1980). The white pock mutants of rabbit poxvirus. I Spontaneous host range mutants contain deletions. Virology 102, 119-132. PADGETT, B. L., and TOMKINS, J. K. N. (1968). Conditional lethal mutants of rabbitpox virus. III Temperature sensitive (ts) mutants; physiological properties, complementation and recombination. Virology 36, 161-167. SAM, C. K., and DUMBELL, K. R. (1981). Expression of poxvirus DNA in coinfected cells and marker rescue of thermosensitive mutants by subgenonic fragments of DNA. Ann Viral (Inst. Pasteur 132E. 135-150. SCHUMPERLI, D., MCFADDEN, G., WYLER, R., and DALES, S. (1980). Location of a new endonuclease restriction site associated with a temperature sensitive mutation of vaccinia virus. Virology 101,281285.