Recombination between temperature-sensitive mutants of herpes simplex virus type 1

VIROLOQY

68,

h-228

(1974)

Recombination

between Herpes

PRISCILLA AND

Department

of

Virology

Temperature-Sensitive

Simplex

Virus

Type

Mutants

of

1’

A. SCHAFFER, MARY J. TEVETHIA, MATILDA BENYESH-MELNICK

and Epidemiology, Accepted

Baylor November

College

of Medicine,

Houston,

Texas

77025

26, 1973

Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 representing 10 complementation groups were examined for their ability to recombine. Efficient recombination was demonstrated between most mutant pairs in standard two-factor crosses. Mutants which failed to complement each other also failed to recombine or recombined with low frequency. Progeny analysis of putative ts+ recombinants demonstrated that complementing clumps and/or multiploid particles did not contribute significantly to recombinant yields. Using recombination data from crosses between 11 mutants representing 7 complementation groups, a provisional linkage rnarJ has been constructed. The map spans approximately 38 recombination units and is linear. INTRODUCTION

Of t.he DNA-containing animal viruses, efficient recombination and the construction Viruses in which eflicient recombination of a provisional linkage map have been occurs offer several distinct advantages for described for rabbitpox virus (Fenner and genetic and biochemical analysis. Genetic Sambrook, 1966). Recombination has also mapping of virus genomes using standard been observed to occur between mutants of recombination techniques can yield detailed adenoviruses (Williams and Ustacelebi, 1971; information about the organization and reguTakemori, 1972)) papovaviruses (Ishikawa lation of virus functions as demonstrated in bact,eriophage systems (Epstein et al., 1963; and Di Mayorca, 1971), and the polyhedral Edgar et al., 1964). Furthermore, the con- cytoplasmic deoxyribovirus, FV3 (Naegek and Granoff, 1971). That efficient recombinastruction by genetic recombination of mutant tion can occur between strains of herpes strains with specified biochemical charactersimplex virus was first demonstrated b> istics has greatly facilitated studies of the Wildy (195.5). As part of a genetic approach functional relationships between essential and non-essential viral genes (Echols, 1971). to studies of the replicative cycle and oncogenic capabilities of herpes simplex Although detailed genetic analysis of animal virus, we have isolat’ed a series of temperaviruses has been hindered by the technical ture-sensitive mutants of herpes simplex problems peculiar to animal virus systems, virus type 1. The isolation, complementation considerable progress has been made recently in animal virus genetics (Ghendon, 1972). and partial characterization of this series of mutants have been described previously 1This investigation was supported by Grant (Schaffer et al., 1970,1973). The present rrCA 10,893 from the National Cancer Institute, port describes studies of the recombination National Institutes of Health. potential of these mutants, and the construc2 Present address: Tufts University School of tion of a preliminary linkage map of herpes Medicine, Department of Pathology, Boston, Massachusetts 02111. simplex virus type 1. 219 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

SCHAFFER,

220 MATERIALS

AND

TEVETHIA,

METHODS

Cell cultures and media. Human embryonic lung (HEL) fibroblasts were propagated and maint’ained as described previously (Schaffer et al., 1973). Constant-temperature water baths with temperature variations of f0.1” were used for incubation of closed vessels and water-jacketed COZ (5 %) incubators with temperature variations of f0.2” were used for petri plates and 24-well tissue culture panels (Linbro Disposo Trays, Linbro Chemical Co., New Haven, Connecticut’). Viruses and virus assays. The KOS strain (Smith, 1964) of herpes simplex virus type 1 (HSV-1) was used as the wild-type (WT) virus, and permissive and nonpermissive temperatures were 34” and 39”, respectively. The induction, isolation, complementation, and partial biochemical characterization of (2s) mutants the 15 temperature-sensitive used in these studies have been reported elsewhere (Schaffer et al., 1970, 1973). Virus stock preparation (Courtney et al., 1970) and the plaque method of virus assay (Dreesman and Benyesh-Melnick, 1967) have been described previously. Quantitation of physical particles in virus stocks was done by the droplet pseudoreplication method (McCombs et al., 1966). Recombination. Recombination between pairs of ts mutants was carried out using standard two-factor crosses essentially as described by Padgett and Tomkins (1968) for rabbitpox virus. A single strain of HEL cells, strain 638, was used exclusively in all recombination tests. Tube cultures of these cells were infected 24 hr after seeding in all cases. Monolayer cultures of HEL cells in tubes containing approximately 2 x lo5 cells/culture were washed once with Tris (hydroxymethyl)aminomethane phosphate buffer at pH 7.4 (Tris). Triplicate cultures were infected with pairs of mutants, each at a calculated multiplicity of 2.5 plaqueforming units (PFU)/cell of each mutant in 0.1 ml (i.e., a combined multiplicity of 5 PFU/cell in 0.2 ml). In parallel, triplicate cultures were infected with single mutants at a multiplicity of 5 PFU/cell in 0.2 ml. Only virus stocks with physical particle-to-PFU ratios of 150 or less were used to infect cells in an effort to control the multiplicity of

rlND BENYESH-MELSICK

effective gmomes. Simultaneous’ assays of inoculum suspensions were performrd in order to confirm calculated input multiplicities and to ensure approximately equal multiplicities of each mutant. If input inocula varied more than 2-fold from the calculated input, results of tests with these mutants were excluded. After adsorption of virus for 1 hr at 37”, inoculum was decanted, infected cultures were washed twice with 2 ml Tris and 2 ml maintenance medium was added to each culture. Cultures were incubated for IS hr at 34” and harvested by 3 cycles of freezing and thawing. Triplicate suspensions were pooled and clarified by lowspeed centrifugation. Supernatant fluids were frozen at -90”. Immediately prior to assay, fluids were routinely exposed to 30 set of sonication at 10 KC, 5”, to disaggregate virus clumps. Virus yields were assayed at 34’ to determine total plaque-forming progeny and at ts+ recombinants 39” to measure and revertants. The proportion of recombinants in mixed infections was determined by dividing the 34” yield assayed at 39” (recombinants and revertants) by the same yield assayed at 34” (total plaque-forming progeny). This value was multiplied by 2, to take into consideration an equal number of reciprocal double-mutant recombinants, and then by 100 to produce the percent recombination or recombination frequency. Since all mutants used in these studies reverted with low frequency and exhibited only low levels of leak, the yields of single mutant infections assayed at 39” (reversion and leak) were not subtracted from 34” mixed yields assayed at 39”. Thus, the formula used for determination of recombination frequencies (RF) was as follows :

RF

=

Yield (A X B)s40 assayed

at 39”

Yield (A X &do assayed

at 34” x 2 x

1 100

Tests of putative recombinants in mixed yields. (a) E$ect of so&cation and jdtration. Both the yield of a mixed infection and an artificial mixture of a mutant pair were used in these tests. The mixed yield was prepared as described under “Recombination” above.

HECOMBINATION

BETWEEN

Th(i artificial mixture was prepared by incubating a mixture containing 1.0 X 10’ PFU/ml of each mutant at 37” for 2 hr to permit clumping. Clumping was further enhanced by centrifugation of the mixture at 1000 rpm for 20 min at 5’ followed by agitat,ion on a Vortex mixer to resuspend clumps. Both the mixed yield and the artificial mixturcl we’re assayed at 34” and 39” (1) before sonication, (2) after 30 set of sonicat’ion at 10 KC!, and (3) after 30 set of sonication followed by filtration through a 450-nm Nillipore filter pretreated with calf serum. (h) Plating eficiency of WT virus and putative is+ recombina,~ts at 34” and 39’. WT virus yields and mixed yields from mutant crosses ~NYL plated at 39”, individual plaques were picked, plaque material was suspended in 0.5 ml of cold Tris containing 1 %I fetal calf serum, and the suspension was sonicated at 10 KC for 30 see to disaggregate clumps. One-t,cnth milliliter of suspension was used to inoculnt’c each of 4 HEL monolayers in 60-mm platts. Two plates were incubated at 34” and two at 39”, and plaques were scored aftor 5 days. Those plaque isolates which produced a combined tot,al of fewer than 10 plaques at’ 34” and X9” wprc excluded from this analysis. RESULTS

Mutallts Used in Recomkation

Analysis

Fiftnen ts mutants belonging t’o 10 complementat’ion groups were tested for their abi1it.y to recombine at 34”, the permissive temperature. Table 1 describes the properties of the viruses and virus stocks used in these studies. Five of the 1.5 mutants studied, belonging to 3 complementation groups, were unable to svnthesize viral DNA at 39” while the rclmai&g 10 mutants, belonging to 7 complementation groups, were able to synthesize viral DNA at this temperature. As seen in Table 1, plating efficiencies (39”/34”) of the mutant’ stocks ranged from 1 X 1O-4 (tsl9u) to 1 X lo-” (ts2lu and ts4b). Although revertants were produced by all mutants, the reversion frequencies were quite low, as seen in the plating efficiencies of the mutants. It is widely recognized that’ physical particle-t,o-PFU ratios of herpes simplex virus may range from about 10, at

HSV

‘121

fs MUTANTS TABLE

1

PROPERTIES OF VIRUSES END VIRUS STOCKS Useu

IN RECOMBIKATION ANALYSIS Couple-

mentation groupa -_ A B C E F (: J M N 0 ___~

Virusa

Wild-type ts15g tS16g t&b ts21u ts4b ts5b i&b ts17g lSl& ts3b ts8b ts12g f.clQu 1420~ /s22u

Vilal DNA

pheno-

tvpe” 390 + _ _ _ _ +

-

+ + + + + + + + +

Propertiesof virus stocks EOPb(39’/3~‘) PP/Prq’ 1 6 6 3 1 1

x x x x X

IO-6 10-S 10-c IO-” lo-”

7 7 8 4 6 2 -1 1 8 5

x 10-e x 10-G x 10-c x 10--G X 10e6 x 10-e x IO-” x 10-J x 10-6 X 10-6

____._____

..~~~

1X 28 7-1 ix 5-l 25 94 113 33 28 150 7!) 14 20 75 33

a Complementation

groups of mutants and viral DNA phenotypes at 39” were determined as previously described (Schaffer et al., 1973) ; lower-case letters g, b, and u after mutant number designate mutagens, NTG, BrdUrd, and UV light, respectively, used for mutant derivation (Sehaffer et trl , 1973). * Virus stocks were grown at 34” and assayed at 34” and 39”. EOP = (PFU/ml39”)/(PFU/ml34”) ; values represent the average of 4-6 separate determinations. c PP/PFU = physical particle-to-PFU ratios of virus stocks. Quantitation of virus particles in stocks was done by the droplet pseudoreplicatic,n method of McCombs et al. (1966).

best, to over 100, depending upon the conditions of virus growth. Most mutant stocks exhibited particle-to-PFU ratios greater than that observed in WT virus stocks (Table 1). Higher ratios were found COIIsistently in stocks of some mutant’s, r.g., t&b and ts6b, and appear to bc charact’eristic of mutants belonging to certain complemrntation groups. Since the effective gene dose of viruses used in genetic studies may t)cx reflected better by the number of physical virus particles which may contain a functional genome rather than by the number of PFU in a virus stock, virus stocks with particle-to-PFU ratios nearest to unity w(‘rf used.

222

SCHAFFER, TEVETHIA, AND BENYESH-MELNICK TABLE 2 RECOMBINATION

Complementation WJUP

Re+Wsion’ frepen&s 9 of mu;ants in selfcrosses

Mutant

2

16

1 PJDC

tsl6g

0.028

PJD

t&b

0.002

-

ts21u

0.004

C

tslb

0.019

E

tslb

0.003

t&b

0.092

ts17g

0.014

tag

0.001

ts3b

0.003

ts6b

0.002

J

ts12g

0.008

M

ts19u

0.022

N

ts2ou

0.026

0

ts22u

G

21 -

0.004

F

MUTANTS

OF HSV-1

-

ts15g

B

ts

Recombination frequenciesb from crows between ts mutants

_ A

BETWEEN

(D

4

_ I

5

6

.-

--

gD

8.! rlD 18.3 t1.2 ! :kO.! 1.11 29.1 20.1 t5.i I kO.[i :*7., 20. I 56. k2.1I *3. 0.f 3 k0. [L

-

18

17

--

3

_-

ND

15.2 t1.9 ND 15.1 to.6 19.4 38.2 :*2.2 t3.2 20.7 33.8 :52.2 k3.9 1.5 14.2 :zto.4 k2.2
12.2 ztl.4 9.8 f2.3 35.5 kO.6 36.9 ztl.1 17.9 ztl.3 16.8 f3.5 14.4 zk2.9 0.0 4 fO.0 4

-

8

12

19

12.3 f0.7 9.0 +0.8 38.7 ztO.5 36.1 ~3.6 9.2 f0.7 19.0 ~3.9 20.5 f3.0 3.2 f1.3 1.6 ho.8 13.5 l 3.4 15.0 ~~2.8 -

ND

?iD

ND

ND

2.0 to.1 26.7 a.5 28.2 k1.8 9.2 k1.3 i15.6 ho. 6 5.3 hO.2 8.6 1l1.4 10.8 kl.5 -

YD

7.5 f0.3 14.8 fl.6 33.2 f1.0 3.4 fl.O 0.3 ho.3 13.2 f3.4 2.6 zto.4 2.4 zt0.5 23.1 f4.1 5.9 f2.1 11.1 zk2.1

ND

5.0 f0.6 34.5 fl.1 32.0 ztl.6 3.2 f0.5 14.0 f2.5 12.4 h3.0 2.0 ztl.2 3.8 fO.9 6.6 f0.3 7.3 l 0.4 4.3 fl.1 31.4 f6.0 22.6 zt0.6 -

25.9 k2.7 24.6 ~t3.3 3.4 h1.1 5.5 fl.5 5.4 *0.7 9.3 l 1.3 11.1 f3.8 0.9 f0.5 -

29.9 k2.4 31.8 zk2.7 4.6 zt2.0 9.7 f1.8 11.1 f2.8 6.1 f2.2 7.4 zt2.1 2.4 *1.0 3.8 fl.1 7.3 zt2.6 41.1 f6.7 -

-

0.005

-

-

22

. --__

-__ND

20

-

* Reversionfrequency = percent wild-type virus in yields from self-crosses; mean values were calculated from all tests with each mutant. b Recombinstion frequency = [yield (A X B)w =-=yed at 39”1 [yield (A X B)w aaaayed at 34”l ’ a ’ ‘O”’ c ND, not done.

Recombination The recombination frequencies obtained in mutant pairwise crosses are presented in Table 2. The data presented in this table represent the mean and standard deviation of 3-5 separate determinations of each cross. The reversion frequencies of mutants in selfcrosses are also presented; these values ranged from 0.001% to 0.028 %. Recombination frequencies ranging from less than 1% (e.g., ts5b X ts6b, tsl7g X tslSg, and k3b X t&b) to greater than 50% (ts21u X k4b) were reproducibly found. Mutants belonging to the same complementation group either failed to recombine (e.g., ts5b X ts6b) or they recombined with low frequency (e.g., ts2b X ts2lu), suggesting in t.he latter

case that the mutations are in the same cistron but affect different sites within the cistron. The reason for the low frequency of recombination found between two complementing mutants, ts5 and ts19, is presently unknown. In selecting mutants for recombination analysis, it was decided to include mutants in complementation groups with more than one member, since mutants in the same group would serve as internal controls. Indeed, it can be seen in Table 2 that mutants defective in the same cistron recombined with similar frequencies in tests with other mutants. Thus, recombination data confirm the assignment of mutants to cistrons as determined by complementation analysis.

RECOMBINATION

Progeny

BETWEEN

Testin<

Although the data presented in Table 2 suggest that recombination occurred with high eficiency in this system, the possibility existed that plaque formation in mixed yields assayed at 39” resulted from complementing clumps of mutants or from complementing multiploid particles. It has been demonstrated that clumps of ts mutants and/or multiploid particles cont’aining two or more genotypically different ts mutants can produce plaques at the nonpermissive temperature by complementation (Simon, 1972). Experiments were therefore performed to test whether plaques formed at 39” were indeed the result of ts+ recombinants. In an experiment designed to determine whether clumping contributed significantly to recombinant yields, a mixed yield of ts2b and tsl9u and an artificially clumped mixt’ure of these mutant’s were subjected to sonication combined with ultrafiltration, as described in Materials and Methods. Such treatment was found previously by Wallis and Melnick (1967) to provide optimal conditions for removal of virus aggregates. As seen in Table 3, plaque formation by the mixed yield assayed at either 34” or 39” was not significantly altered by sonication or by combined sonication and filtration. Alt’hough combined sonication and filtration reduced totma infectivity by approximately

22:t

HSV fs MUTANTS

30 %, a similar 30 % reduction in ts+ plaqucb$ was also observed. Thus, the recombination frequency was not affected by this treatment. The recombination frequency obtained in this cross (last column of Table 3) agrees wall with previous determinations (Table 2). Thc~ “recombination frequency” calculated for t’he untreated artificial mixture was 0.06 ?;‘. as would be expected of a mixture of IS mutants. However, unlike the results ob tained with the mixed yield, sonication and combined sonication and filtration decreased this value by 4- and &fold, respectively. These results suggested that a small numh(br of complementing ts mutant virus clumps were present in the artificial mixture. Although ts+ recombinants in the mixed yield did not appear to arise from complcment~ing clumps, all virus suspensions were routincal? sonicated immediately prior to assay to minimize clumping. Additional evidence which demonstratt,r that plaques formed by mixed yields at 39”’ were true ts+ rccombinants and did not arise. from complementing clumps or multiploid particles is shown in Table 4. Yields from 7 mutant crosses and from WT virus w(lrc plat’ed at 39” and plaques were picked and replated at 34” and 39” as described in Materials and Methods. Upon replating, 7:j of the 74 plaques from t,he 7 crosses plat,ed in a manner equivalent to the 10 WT control.

TABLE

3

EFFECT OF SONICATION AND FILTRATION ON PLAQUE FORMATION OF A MIXED RECOMRINATIOS YIELD AND AN ARTIFICIAL MIXTURE OF MUTANTS ts2h AND tsl9u Preparation

Treatment

PFU/ml 34”

Mixed yieldfi (/s2b X ts19u) Artificial mixtureb (ts2b + tsl9u)

None Sonicated Sonicated None Sonicated Sonicated

and filtered

and filtered

2.8 2.6 2.0 5.5 6.5 9.0

X x x x X x

at: 39”

10’ 10’ 101 106 lo6 106

-

2.2 x 106 1.9 X 106 1.6 X lo6 1.5 X 103 5.0 x 102 5.0 X 102

Recombination frequency PFU/ml39” [ PFU./ml34”

1 x2xloo 15.8 14.6 16.0 0.06 0.01ti 0.012

a The mixed yield consisted of supernatant fluids (centrifuged at 1000 rpm, 15 min, 5”) from threetimes frozen and thawed cell cultures mixedly infected with ts2b and tsl9u. b The artificial mixture was prepared as described in Materials and Methods from supernatant fluids of cell cultures infected with each mutant alone. Infected cultures were frozen and thawed three times: and clarified by centrifugation at 1000 rpm, 15 min, 5”, as for the mixed yield.

SCHAFFEIZ, TEVETHIA,

224

AND BENYESH-MELNICK

TABLE 4 PLATING

EFFICIZNCIES

OF WILD-TYPE

VIRUS

AND PLAQUE

PLATED Plaque number

FROM RECOMBINATION

PROGENY

Plating efficiencies

_ Y

Putative recombinant plaques produced in crosses between

Wild-type ,irus plaques

--

34”

T

39”

f slZgXts2b _

-

34”

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

ISOLATES

AT 39”O

35 20 20 80

18 33 45 80 75 20

25 12 17 70 25 34 37 120 80 20

10 20 40 20

15 25 25 12

12

39” __.

11 30 20 20 21 18 19 B 18

slZgXf~6b 34”

39”

11 150 18 16 35 18 8 6 18

14 210 13 11 38 14 12 5 14

slZgxIs3b 34”

390

21 17 14 18 10 33 80 130 10 15

15 16 13 25 11 34 110 150 13 12

T

IS2OUX ts 17g 340 90 20 23 20

11 60 60 14 11 20

fs 22u x

-

390

340

110 30 21 30 8 30 100 17 13 20

21 9 14 5 13 150 23 4

6

Is

17g

390

11

22 10 16 6 18 170 17 6

I, s

8b X fs 18g

_-

-

34” 5

10 8 19 7 13 110 18 15 34

39”

5 8 12 14 8 20 80 13 11 38

s

2b X fs 8b

340 60 60

18 13 80 13 60 23 21 80 I1 8 11 35 15 8 16

I-

-

390

40 48 16 19 40 15 70 32 26 50 14 10 9 37 12 6 17

i a Wild-type virus and mixed yields from mutant crosses were plated at 39” and individual plaques were picked. Plaque material was suspended in 0.5 ml Tris containing 1% fetal calf serum and the .suspension was sonicated at 10 KC for 30 sec. Four HEL monolayers in 60-mm plates were inoculated with 0.1 ml each of suspension. Two plates were incubated at 34” and two at 39” and plaques were counted after 5 days. plaques at 34” and 39”, and thus behaved true ts+ recombinants.

as

The Genetic Map From the data presented in Table 2, a provisional linkage map of mutants belonging to 7 complementation groups has been constructed (Fig. 1). The map spans approximately 38 recombination units; the longest interval is 38.7 units and the sum of the shortest intervals, 37.2 units. So far, the map appears to be linear with no evidence of circularity. Although recombination experiments were conducted with 15 mutants, data involving only 11 of these mutants were used in the construction of the linkage map. Since tests of tsl5g and tsl6g had not been completed with certain other mutants at the time of t’his writing, these mutants were not included in mapping attempts. Their available

recombination frequencies were included in Table 2 only to demonstrate that additional DNA- mutants recombined efficiently. Two mutants, however (ts4b and tslgu), yielded recombination frequencies which were not additive with other mutants and therefore could not be positioned on the map. The reason for the nonadditive values obtained with these two mutants is not known. DISCUSSION

Recombination studies with 15 ts mutants of HSV-1 have demonstrated that tsf recombinants were produced efficiently in many pairwise crosses. The recombination frequencies obtained were reproducible and characteristic for each mutant pair. Mutants which failed to complement each other recombined with a very low frequency, demonstrating that mutants defective in the

IiECOMBINATION

BETWEEN

HSV

Is MUTANTS

FIG. 1. Linkage map of HSV-1. Eleven ts mutants representing 7 complement&ion groups were ordered by standard two-factor crosses. Recombination frequencies presented are the average values of from 3--5 determinations of each cross. The data used to construrt the map were taken from Table 2.

same cistron do not recombine efficiently. Since certain cistrons were represented by more t’han one mutant in t’hese tests, and since each mutant was independently derived (i.e., the mutants were not “sisters”), the observation that mutants defective in the same cistron recombined with other mutants

with approximately equal frequency lends further support to the validity of the rccombination values obtained. The use of mutant stocks with opt’imal plating efficienties and particle-to-PFU ratios very likely contributed to the reproducibility of the recombination frequencies obtained, as did

226

SCHAFFER,

TEVETHIA,

the use of a single strain of HEL cells at a common time after seeding for all recombination tests. As shown in Table 3, complementing ‘clumps occurred only minimally in this system. Analysis of progeny from recombination yields (Table 4) supports this finding -and demonstrates that complementing multiploid particles, if present, were also rare. In .electron microscopic studies of herpes simplex virus, the percent of multiploid virions was found to be approximately 1% (Darlington and Moss, 1968; Nii et al., 1968). ‘Therefore, only in crosses which produced low levels of recombinants (e.g., crosses within complementation groups) could such levels of complementing clumps and/or multiploid particles significantly alter the recombination frequencies observed. The linkage map of HSV-1 was con..structed from the average of multiple tests of all pairwise crosses. Six of the 7 cistrons ,ordered fall within an interval of about 20 recombination units. The seventh cistron falls 20 units away from its nearest neighbor cistron and is thus only loosely linked to all other cistrons ordered. Although variations were observed in recombination frequencies from test, to test, as seen from the standard deviations of frequencies (Table 2), both the ,order of mutants and the relative distances between them were consistent. In addition, the observation that independently derived mutants defective in the same cistron mapped in the same region lends further .support to the order of mutants obtained. The failure of some mutants (ts4b and .tsl9u) to recombine additively with other mutants suggests the possibility that these mutants may be doubles. Efficient recombination and the construction of a linkage map have been reported by Brown et al. (1973) using three-factor crosses. In their study, however, 8 of the 9 cistrons mapped fell within a distance of from about 5 (longest interval) Do 8.5 (sum of the shortest intervals) recombination units. Furthermore, the intervals between certain cistrons were found to be less than one recombination unit. The use of three-factor crosses in the study of Brown et al. (1973) thus reflects a unique advantage of such crosses, i.e., for the ordering of cistrons re-

AND

BENYESH-MELNICK

liably over short intervals. Two-factor crosses were probably not sufficiently sensitive to resolve the order of mutants over very short distances in our system, i.e., within the same cistron. However, the reproducibility of frequencies obtained over intervals of approximately 2 recombination units (the shortest intercistronic interval on our map) or more, was high. Our map presently spans about 38 units and, although there is no evidence of circularity, both the length and topology of the map may change as additional markers are ordered. The functional organization of the HSV-1 linkage map cannot be evaluated because of the small numbers of genes which have been ordered. Also, the extent to which the cistrons ordered in the study of Brown et al. (1973) and in our study overlap awaits further functional and genetic characterization of both sets of mutants. It is of interest, however, that in our studies the DNAmutants in complementation group B are found at the left-hand end of the map, and mutants in complementation groups F and G, in the center of the map, possess a temperature-sensitive structural component of the virion as determined in thermal inactivation experiments (Schaffer et al., 1973). Similar functional clustering of cistrons on the map of Brown et al. has also been observed (J. H. Subak-Sharpe, personal communication). Clearly, from the work of Wildy (1955), Brown et al. (1973), and the present study, highly efficient recombination does occur in the HSV-1 system. Certain features of the HSV replicative cycle may help to explain this phenomenon. The localization of viral DNA synthesis within the nucleus of infected cells provides optimal opportunity for genetic exchange. The physical nature of the HSV DNA molecule is a second feature. The molecule is relatively large (90-100 X lo6 daltons; Kieff et al., 1971; Graham et al., 1972) ; it is double-stranded and linear. These features make it an ideal molecule for frequent genetic exchange. Furthermore, alkali denaturation experiments have indicated that HSV DNA contains single-strand interruptions or alkali-sensitive bonds at unique sites within the molecule (Kieff et al., 1971; Frenkel and Roizman, 1972; Gordin

RECOMBINATION

BETWEEN

et al., 1973). The presence of single-strand interruptions in the DNA of bacteriophage T5 has also been demonstrated (Davison et al., 1964). That’ such labile sites within the HSV DNA molecule represent preferred sites of breakage and reunion, as has been suggestcd for T5 (Lanni et al., 1966), remains to be determined. ACKNOWLEDGMENTS We thank

Mrs.

Mr. R. T. Lewis, excellent

technical

S. Combs, Miss B. J. Nowak, and Mr. R. Striker for their assistance.

REFERENCES

BROWN, S. M.,

RITCHIIG, D. A., and SUBAKSHARPE, J. H. (1973). Genetic studies with herpes simplex virus type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Viral. 18, 329-346. COURTNI~Y, R. J., MCCOMBS, R. M., and BXNYESHMELNICK, M. (1970). Antigens specified by herpesviruses. I. Effect of arginine deprivation on antigen synthesis. Virology 40, 379386. DARLINC:TON, R. W., and Moss, L. H. III. (1968). Herpesvirus envelopment. J. Viral. 2.48-55. DAVISON, P. F., FRFXFELDER, I)., and HOLLOWAY, B. W. (1964). Interruptions in the polynucleotide st,rands in bacteriophage DNA. J. Mol. Biol. 8, 8-10. DRI~ESMAN, G. II., and BENY~XH-MELNICK, M. (1967). Spectrum of human cytomegalovirus complement-fixing antiFens. J. Immunol. 99, 110&1114. ECH~LS, H. (1971). Regulation of lytic development. 1~ “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 247-270. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. EDGAR, R. S., DENHARDT, G. H., and EPSTEIN, R. II. (1964). A comparative genetic study of condit,ional lethal mutations of bacteriophage T4D. Gerretics 49, 635-648. EPSTEIS, 1:. H., BOLLE, A., STEINB~;RG, C. M., KELLI,:NBE:RG~:R, E., BOY DF: LA TOUR, E., CHEVALLXY, It., EDGAR, R. S., SUSMAN, M., DENHARDT, (;. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quad. Riol. 28, 375-394. FENNER. F., and RAMBROOK, J. F. (1966). Conditional lethal mutants of rabbitpox virus. II. Mutants (p) that fail to multiply in PK-2a cells. virology 28, tioo409. FRI’.NKXL, N., and R~IZMAN, B. (1972). Separation of the herpesvirus deoxyribonucleic acid duplex into Irnique fragments and intact strand on

227

HSV fn MUTANTS

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