Hydroxylamine mutagenesis of HSV DNA and DNA fragments: Introduction of mutations into selected regions of the viral genome

VIROLOGY

98, 168-181(1979)

Hydroxyla-mine Mutagenesis of HSV DNA and DNA Fragments: introduction of Mutations into Selected Regions of the Viral Genome CHIEN-TS CHU,* DEBORAH S. PARRIS,? RICHARD A. F. DIXON,“~~ FLORENCE E. FARBER,S AND PRISCILLA A. SCHAFFER’+,’ *Department of Virology, Carleton College,

Baylor College of Medicine, Houston, Texas Minnesota 55057; and tThe Sidney

North&d, Harvard

Medical

School, Accepted

Boston, June

Massachusetts

i’RX30; $Department of Biology, Farber Cancer 02115

Institute,

28, 1979

Procedures for the induction and isolation of temperature-sensitive (ts) mutants by in mutagenesis of purified HSV DNA and DNA fragments have been developed. In order to establish conditions for the mutagenesis of viral DNA fragments with hydroxylamine (HA), virion-associated and intact DNA were mutagenized first. Under the conditions of mutagenesis employed, HA was shown to be a more powerful mutagen of purified DNA than of virion-associated DNA. Mutagenesis of intact DNA resulted in the identification of three new complementation groups. A mixture ofEcoR1 fragments A and B was next mutagenized. Mutations in DNA fragments were recovered by cotransfection with intact, wild-type DNA. Six ts mutants were isolated from the progeny of the mixed infection. Five of these mutants were analyzed further. One mutant, ts508, failed to complement tsB2 which had previously been shown to lie in fragment B. Like tsB2 and other members of this group, ts508 is DNA-. The remaining four HA-induced mutants constitute three new DNA+ complementation groups. Marker rescue experiments indicate that mutants in two of these groups lie in fragment A. vitro

INTRODUCTION

Herpes simplex virus (HSV) DNA is a linear double-stranded molecule of molecular weight 97-99 x lo6 (Kieff et al., 1971; Graham et al., 1972). In addition to its large size, HSV DNA is structurally complex in that it exists in four forms which differ in the relative orientation of the two unique regions of the genome, L and S, to each other (Sheldrick and Berthelot, 1974; Wadsworth et al., 1975; Delius et al., 1976). Although the total number of genes encoded by HSV DNA is not known, complementation analysis of temperature-sensitive (ts) mutants has led to the identification of 23 complementation groups of HSV-1 and 20 of HSV-2 (Schaffer et al., 1978). Physical and 1 Author to whom reprint dressed.

requests should be ad-

0042~6822/79/130168-14$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

genetic mapping studies with available mutants have demonstrated that large regions of the viral genome are currently mutationally silent. Furthermore, Morse et al. (1978) and Marsden et al. (1978) have recently mapped the DNA templates specifying 26 viral polypeptides by the analysis of recombinants between HSV-1 and HSV-‘2. Although the locations of the templates specifying these polypeptides have been determined, very little is known of their functional roles in the viral replicative cycle. Identification of the functional role of each polypeptide will require the isolation of mutants in the corresponding gene. Ultimately, a complete understanding of the structural and functional organization of the HSV genome will necessitate the identification of all viral genes; therefore, the ability to introduce mutations into specific DNA sequences known to encode individual viral 168

MUTAGENESIS

OF HSV

polypeptides and into hitherto mutationally silent sequences would clearly benefit both the genetic and biochemical analysis of HSV. This report describes a simple, rapid procedure for the introduction of mutations into preselected regions of the HSV-1 genome. The identification, localization, and preliminary characterization of ts mutants representing six complementation groups is also presented. The technique should be applicable to a wide range of systems in addition to HSV, i.e., mutations could be introduced into selected regions of any DNA species. MATERIALS

AND

METHODS

Cells and cell cultures. Serially propagated human embryonic lung (HEL) fibroblasts, primary rabbit kidney cells (RK), and African green monkey kidney cells (Vera) were used in this study. Cells were grown in Eagle’s minimum essential medium (Auto-Pow; Flow Laboratories, Rockville, Md.) containing 10% fetal bovine serum, 0.03% glutamine, and 0.225% NaHCO,. Cells were maintained in the same medium containing 5% fetal bovine serum. Virus stocks were grown in HEL cells, viral DNA was prepared from virus propagated in RK cells, transfection and marker rescue experiments were carried out in RK cells, and Vero cells were used for assays of viral infectivity. Virz~ growth and assay. The KOS strain of HSV-1 was used as the wild-type virus. The isolation, complementation, and genetic mapping of ts mutants have been described previously (Schaffer et al., 19’73, 1974). ts mutants in complementation groups A through P were used in complementation tests with new ts mutants. Mutants in groups A through P are members of the standard set of complementation groups 1. - 1 through 1. - 15 as reported by Schaffer et al. (1978). The origin of mutants representing complementation groups l.-16 through l.-23 has been reported previously (Schaffer et al., 1978). Virus stocks were prepared and assayed as described (Schaffer et al., 1978). The permissive temperature was 34” and the nonpermissive temperature, 39”.

DNA

FRAGMENTS

169

Hydroxylamine (HA) mutagenesis oj’ HSV virions. Mutagenesis of HSV-1 with HA was conducted essentially as delineated by Freese et al. (1962) and Robb et al. (1970). Virus (0.2 ml, 1 x lo* PFU/ml) which had been plaque-purified three times was mixed with 1.8 ml of 2.0 M HA (Eastman Chemical, Rochester, N. Y.) in Trisbuffered saline. Virus mutagenized with HA at 37” for various lengths of time was diluted 1:lOO in cold maintenance medium and was assayed immediately in Vero cells. For mutant isolation, virus that had been inactivated for a period of time which resulted in 16% survival of infectious virus was passed in 10 replicate tube cultures of HEL cells in order to resolve mutational heterozygotes. Cultures were frozen when cytopathic effects were generalized and infected cell suspensions were frozen, thawed, sonicated, and clarified by low speed centrifugation. The resulting virus suspensions were assayed at 34” in Vero cell monolayers and plaques were picked and screened for temperature sensitivity at 34 and 39”. Zsolation of HSV-I DNA. HSV-1 DNA was extracted from partially purified virions as previously described (Farber, 1976). Briefly, monolayers of RK cells were infected with either the wild-type strain of HSV-1 or a ts mutant derived from strain KOS at a multiplicity of 0.01-0.1 PFU/ cell. The virus was allowed to adsorb for 1 hr at 37”. The inoculum was removed and maintenance medium was added. Infected cultures were incubated at 34” until all cells exhibited generalized cytopathic effects (3-4 days). Cells were then scraped into the medium and pelleted by centrifugation at 600 g for 15 min. The medium was decanted and held on ice for later use. The cell pellet was resuspended in 5-10 vol of RSB (0.01 M Tris-HCl, pH 7.4, 0.01 M NaCl, 0.003 M MgCl,) and allowed to swell at 0” for 10 min. Cells were ruptured by four to six strokes in a tight fitting Dounce homogenizer. The nuclei were removed by centrifugation at 1100 g for 15 min. The cytoplasm was carefully removed and added back to the original cell culture medium. Virus was pelleted by centrifugation at 12,000 g for 1 hr in a Sorvall GSA rotor at 4”. The supernatant fluid was discarded,

170

CHU ET AL.

and the virus pellet was resuspended in a small volume of TNE (0.01 M Tris-HCl, pH 7.4, 0.1 M NaCl, 0.001 M EDTA). The virus was layered over a ZO-60% sucrose gradient in TNE and banded by centrifugation at 112,000 g for 1 hr in a Spinco SW27 rotor. The light scattering band in the middle of the tube was collected by bottom puncture and resuspended in -35 ml of TNE. Virus was pelleted at 40,000 g for 15 min in a Spinco SW27 rotor and resuspended in a small volume of TNE. Virions were lysed by the addition of 10% SDS to a final concentration of 1% and the suspension was rocked gently. The DNA solution was then diluted 1:4 with TNE and 5.0 M sodium perchlorate was added to a final concentration of 1.0 M. The DNA was deproteinized two to three times by extraction with an equal volume of chloroform: isoamyl alcohol (24:l). The DNA was dialyzed with two to three changes against 50 vol of 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2). DNA was concentrated by dialysis against solid polyethylene glycol (Carbowax, PEG 6000), and the PEG was removed by dialysis overnight against 0.1 x SSC. Aliquots of DNA were frozen at -70”. Restriction enzyme digestion and agarose gel electrophoresis. Restriction enzymes were purchased from Miles Laboratories (EcoRI) or New England Biolabs (XbaI, NindIII). DNA was digested as follows: 30-50 pg of HSV-1 DNA was mixed at a ratio of 2 units of enzyme/&g of DNA in either 0.1 M Tris-HCl, pH 7.5, 0.01 M MgC&, 0.02 M NaCl forEcoR1 and Hind111 or 0.006 M Tris-HCl, pH 7.9, 0.006 M 0.006 M 2-mercaptoethanol for M&L, XbaI. The reaction was allowed to proceed at 37” for 2 hr at which time it was stopped by the addition of one-fourth volume of 1% SDS, 0.05 M EDTA, and 0.1% bromphenol blue in 60% glycerol. Samples were heated to 65” for 10 min prior to electrophoresis to eliminate aggregates. Fragments were separated by electrophoresis on 0.4% agarose (Low EEO agarose, Sigma Chemical Co., St. Louis, MO.) gels. Fragments were separated on horizontal slab gels 15 x 25 x 0.5 cm in E-buffer (0.04 M Tris-acetate, pH 8.0, 0.005 M

sodium acetate, 0.001 M EDTA) at 1.5 V/cm until the dye band reached the bottom of the gel (-18 hr). Gels were stained with 1 pg/ml ethidium bromide, and fluorescent bands were visualized under uv light. Bands were excised from the gel and crushed. The DNA was allowed to diffuse out of the gel overnight at 4”. Agarose was removed by centrifugation at 12,000 g for 30 min in a Sorvall SS-34 rotor and DNA was precipitated by addition of sodium acetate to 0.5 M and 2.5 vol of absolute ethanol. After standing at -20” overnight, the DNA was pelleted at 12,000 g for 30 min and air dried. The pellet was resuspended in a small volume of distilled water and made 1.0 M in sodium acetate. Ethidium bromide was removed by extraction with isoamyl alcohol. The DNA was again precipitated by the addition of 2.5 vol of ethanol and allowed to stand overnight at -20”. To insure purity, individual fragments were reelectrophoresed on a 0.4% agarose gel and isolated as described above. Mutagenesis of intact HSV-1 DNA and fragments. A fresh solution of HA in 0.1 M Na,P,O,, 0.002 M NaCl was prepared and adjusted to pH 6.0 with NaOH. HSV-1 DNA (-100 pg, containing 1 x lo5 PFU) in 0.1 x SSC was mixed in equal volumes with HA solution which had been diluted 1:20 in 0.1 x SSC to yield a final concentration of 0.1 M prior to addition to the DNA. The mutagen was allowed to react at 70” for the times indicated. The reaction was stopped by the addition of a ZO-fold excess of 0.1 x SSC containing 10% acetone, and mutagenized DNA was stored at -70”. Marker rescue and marker transfer. The same procedure was used both for the transfer of mutations from mutagenized fragments to intact DNA and for marker rescue of ts mutants. Restriction enzyme-generated fragments, purified as delineated above, were mixed with a quantity of wildtype or mutant DNA equivalent to 500 PFU of intact DNA in HBS (0.002 M HEPES, pH 7.05, 0.14 M NaCl, 0.005 M KCl, 0.0005 M Na,HPO,, 0.006 M dextrose); normal RK cell DNA was added to give a final DNA concentration of 20 pg/ml. RK cell DNA was nrenared as nreviouslv de-

MUTAGENESIS

OF HSV DNA FRAGMENTS

171

mutagen, 5scribed (Farber, 1976). CaCl,, 2.0 M was duced by the thymine-specific added to a final concentration of 0.125 M bromodeoxyuridine (BUdR) (Schaffer et al., and the DNA was allowed to precipitate at 1978). Because the principal mutagenic acroom temperature for 20 min. During the tivity of HA is the induction of cytosinetransitions and because HSV incubation period, monolayers of RK cells to-thymine DNA is G:C rich, it was felt that HA might were trypsinized and suspended in growth medium. For each sample, two million cells induce mutations in genes other than those were added to 15-ml conical centrifuge favored by BUdR mutagenesis. Furthertubes. The cells were pelleted at 180 g for more, HA acts on isolated nonreplicating 5 min and the medium decanted. The cell DNA as well as on DNA packaged within pellet was then resuspended in the small virions (Freese et al., 1961; Freese and volume of medium remaining in the tube Strack, 1962; Budowsky, 1976). (-0.1 ml). One milliliter of DNA suspenHA has two distinct effects on virus parsion was added to cells and they were in- ticles: one involves the inactivation of infeccubated at 37” in a water bath with vigorous tivity by direct interaction with the virus shaking for 45 min. After incubation, an capsid and the other is a mutagenic effect additional 2 x lo6 RK cells in 10 ml of on the genome (Freese et al., 1961). Congrowth medium were added to each tube. ditions were therefore selected which would Cell suspensions were plated in duplicate minimize the nonmutagenic inactivation of 60-mm petri plates and cells were allowed to HSV. Because high concentrations of HA attach to plates at 34” for 4 hr; medium protect virus particles from nonmutagenic was then removed and new growth medium inactivation (Freese et al., 1961>, 1.8 M added. Incubation was continued for an ad- HA was selected for mutagenesis of intact ditional 12 hr at which time the medium virions. When virions were treated with HA was changed again. Infected cells were and sampled at lo-min intervals for 1 hr, incubated at 34” until all cells exhibited inactivation was found to be linear through cytopathic effects (4-5 days). Cells were 30 min, at which time 22% of viral infecharvested by scraping into the medium and tivity remained (Fig. 1). Thereafter, only suspensions were frozen at -70”. Cells were moderate inactivation was evident (60 min, then thawed, sonicated for 1 min, and 9% survival). The virus preparation which clarified by centrifugation at 180 g for 15 had been treated with HA for 30 min was min. For marker rescue, virus was assayed passed once to resolve mutational heterozyat both 34 and 39” as described. For frag- gotes, progeny were plated, and plaques ment mutagenesis, virus was plaqued in were picked. Of 458 plaque isolates, 7 exVero cells at 34” and individual plaques hibited some degree of temperature-sensiwere picked. Plaque isolates were grown in tivity. Only one (ts277), however, exHEL cells and virus was assayed for growth hibited a plating efficiency (EOP = PFU at both 34 and 39”. Isolates which grew at 34” but not at 39” were plaque purified three times and stocks were prepared as described above. Characterization of new ts mutants. Complementation tests, recombination analysis, and determination of the viral DNA phenotype were conducted as described (Schaffer et al., 19’73; 1974; Aron et al., 1975). RESULTS

Mutagenesis of Virion-Associated HSV DNA with Hydroxylamine (HA)

More than 80% of the HSV-1 and HSV-2 ts mutants identified to date have been in-

FIG. 1. Kinetics of inactivation of HSV-1 virions by HA. Virions were treated with 1.8 M HA for the times indicated and assayed for infectivity at 34”.

172

CHU ET AL.

39”/PFU 34”) of less than 10e4. This mutant was plaque purified three times and stocks were prepared for further study. HA

Mutagenesis of Intact, Infectious HSV-I DNA HA is mutagenic primarily on cytosine residues in single-stranded DNA (Freese and Strack, 1962). Therefore, purified DNA was subjected to thermal denaturation during mutagenesis. Because high salt inhibits denaturation, mutagenesis was conducted in 0.05 x SSC. HA was used at a concentration of 0.05 M. The inactivation of HSV DNA by 0.05 M HA in 0.05 x SSC at 170 for 60 min is shown in Fig. 2. Greater than 90% of HSV DNA infectivity was lost after 15 min in the presence of HA, whereas infectivity remained unchanged after 60 min in the absence of HA. In order to determine whether HA treatment of intact HSV DNA was mutagenic, RK cells were transfected at 34” with DNA treated for 9, 11, and 13 min. The progeny of this transfection were plated, and plaques were picked and tested for temperature sensitivity. None of 36, 5 of 200, and 12 of 223 plaque isolates exhibited temperature sensitivity when isolates were tested from the 9-, ll-, and 13min samples, respectively. Only 2 of the 5 mutants induced following 11 min of treatment with HA, and 6 of the 13 mutants induced following 13 min of treatment exhibited plating efficiencies of 1O-4 or less. It is not possible to determine the frequency of mutant induction by HA because

FIG. 2. Kinetics of inactivation of infectious HSV-1 DNA by HA. DNA was treated at 70” for the times indicated in 0.05 x SSC with (0) or without (0) 0.05 M HA and-assayed by transfection following precipitation with 0.125 M CaCI,.

mutagenized DNA was passed once before plating. However, it should be noted that a good correlation was observed between the length of treatment, inactivating activity, and mutagenic activity. With increasing time of HA treatment, inactivation of infectivity was more pronounced and the frequency of mutant isolation was greater. Complementation Mutants

Tests with HA-Induced

In order to determine whether HA effectively mutagenized viral genes not already represented by mutants induced with other mutagens, complementation analysis was conducted on all pairwise combinations of the following mutants: (a) ts277, the mutant isolated from HA-mutagenized virions, (b) the eight ts mutants isolated following HA mutagenesis of infectious viral DNA, and (c) mutants representing 15 complementation groups of HSV-1, strain KOS. In tests among HA-induced mutants, ts2’77 and ts401 failed to complement as did mutants ts487, 488, and 494 (Table 1). For the two complementation groups with more than one member, only one mutant in each group (ts401 and ts494) was selected for further testing. The fact that ts487, 488, and 494 failed to complement each other suggests that these mutants may be clonally related -consistent with their isolation from progeny of the same transfected culture. Thus, among themselves, the nine HA-induced mutants represented six complementation groups. In tests between representatives of these six groups and mutants in established groups, three pairs of mutants failed to complement, ts277 and tsB2b, ts201 and tsF18g, and ts449 and tsJ12g (Table 2). In a separate series of tests, ts401 (which did not complement ts277) also failed to complement tsB2b and tsB2lb (data not shown). Therefore, ts27’7 and ts401 were placed in group B and designated tsB27h and tsB32h; ts201 was placed in group F and designated tsF29h; and ts449. was placed in group J and designated tsJ33h according to established procedure for KOS ts mutants (Schaffer et al., 1973). Likewise, ts372 has been designated tsQ26h; ts484, tsR30h; and ts48’7, 488, and 494, tsS38h, 39h, and 31h,

MUTAGENESIS

TABLE COMPLEMENTATION

AMONG

ts MUTANTS

173

OF HSV DNA FRAGMENTS

INDUCED

BY

1

HA MUTAGENESIS

OF VIRIONS

Complementation

AND INFECTIOUS

DNA

index”

ts

mutant

277

201

372

401

HA-treated virions

277

-

20.3

4.2

0.3

HA-treated HSV DNA, 11 min

201 372

-

-

13

-

26 440

HA-treated HSV DNA, 13 min

401

-

-

-

484 487 488

-

-

-

-

-

-

-

494

-

-

-

Source

449

484

449

6.1

4.1

487

488

494

65

19

18

17

42 13

490 26

-

-

510

21

56

210

4.1 39

31

-

32 -

18 6.1

-

-

-

-

-

-

-

-

-

-

-

-

1.0 -

-

-

-

-

-

3.9

7.9

9.6 -

25

7.7 0.3

0.6 -

(1Complementation index = (A + B)&AaP + B 39o,assayed at 34”. Values less than 2 (italicized) were considered to be negative @chaffer et al., 1973). The indices shown represent the average of two or three separate determinations.

respectively. Efforts to include ts48’7 and 488 in tests with previously described mutants were unsuccessful due to excessive levels of leak (t487, ts488) and reversion (ts488) and to the inability to produce sufficiently high titered virus stock. The results of complementation tests have shown that HA mutagenesis has resulted in the identification of three new complementation groups (Q, R, and S) in the series of KOS ts mutants isolated in this laboratory.

Mutagenesis

of HSV-1 DNA Fragments

Having established that 0.05 M HA effectively induced ts mutations in isolated DNA under the conditions of salt concentration and temperature selected for mutagenesis, isolated viral DNA fragments were subjected to mutagenesis. For this purpose, EcoRI fragments A and B were selected. These fragments are of molecular weights 13.7 and 13.5, respec-

TABLE

2

COMPLEMENTATION BETWEEN HA-INDUCED MUTANTS PREVIOUSLY IDENTIFIED COMPLEMENTATION Complementation

AND MUTANTS GROUPS

IN 15

index”

ts

mutant

Al

277

65

HA-treated HSV DNA, 11 min

201 372

2.8 120

HA-treated HSV DNA, 13 min

449 484 494

35 24 5.1

SOWW

HA-treated

B2 1.0

C7

D9

E6

150

140

120

5.6 460

8.9 7200

71 8.2 75

F18

G8

HlO

Ill

512

K13

L14

Ml9

NZO 022

1’23~

220

150

48

2’70

85

62

2.3

320

190

50

14

4.6 2700

0.8 190

4.7 120

3.3 80

12 230

3.3 68

4.6 140

3.9 71

23 730

39 54

28 50

4.2 31

20 5.5 3500

57 6.7 150

10 39 390

27 220 1100

0.2 270 570

6.2 73 320

10 32 92

72 14 730

13 21 220

83 44 150

8.1 2.8 37

virions 7.4 680

38 270 14M)

49 12 1150

u See footnote to Table 1. * Complementation tests with tip23 were conducted dominant lethal effect Of ts&% by lowering the multiplicity

13 230 170

using a modification of the standard test (Sehaffer et al., 1978) designed of this mutant lo-fold relative to that of the other mutant in the pair.

to decrease

the

174

CHU ET AL.

o

b

0.0

0.1 I

E 10.2

0.2 ,

I

0.3 I

A

0.4 I

LOM

0.5 I

F

0.6 I

N

G

0.7 I

D

0.1 1

: I

c I

0.9 I

II

1.0

K

13.7

FIG. 3. EcoRI restriction endonuclease cleavage maps of HSV-I (KOS) DNA (Skare and Summer, 197’7). The two maps represent the P and I, orientations of the genome. The heavy lines indicate the regions of the genome which were mutagenized. Above the scale of fractional distance is the sequence arrangement of the genome (Hayward et al., 1975). The preparative gel on the left shows a primary EcoRI digest of HSV-1 DNA; the gel on the right illustrates reelectrophoresis ofEcoR1 A and B fragments isolated from the first gel.

tively. They corn&-rate in agarose gels, and can be obtained in large quantities (Fig. 3). More importantly, all sequences in fragment A and the sequences in L in fragment B constitute regions of the genome in which no ts mutants of strain KOS are known to be located. The B locus, which lies in sequences

in S in the B fragment, served as an internal control in that isolation of additional group B mutants would help to confirm the specificity of mutagenesis. Although the precise G:C composition of fragments A and B is not known, fragment A lies in an A:T rich region of the genome whereas

MUTAGENESIS TABLE

OF

HSV

3

COMPLEMENTATION AMONG~SMUTANTS INDUCED BY HA MUTAGENESISOFFRAGMENTSA AND B Complementation

index”

ts mutant

508

701

704

797

953

-

6 -

2300 12 -

1500 0.8 700 -

240 38 4 170 -

508 701 704 797 953 (1 See footnote

to Table

1.

fragment B spans the G:C rich joint region (Hayward et al., 1975). Thermal denaturation and mutagenesis of cytosine residues in denatured single-stranded regions would theoretically favor mutagenesis of fragment A. The mixture of fragments A and B was mutagenized with HA for 13 min, the time of HA treatment which had yielded the largest number of mutants in tests with intact DNA. Mutagenized fragments were mixed with intact HSV DNA and RK cells were transfected with the CaPO,-precipitated mixture. The progeny of transfected RK cells which theoretically contained wildtype virus and “rescued” mutants, were plated, plaques were picked and ts mutants were identified. Five ts mutants which exhibited plating efficiencies of 10e4 or less were used in further tests. Complementation Tests with Mutants Derived from Mutagenized Fragments Complementation tests among the five mutants demonstrated that two, ts701 and TABLE

DNA

175

FRAGMENTS

ts797, failed to complement one another (Table 3). Thus, the five mutants represented four complementation groups. Representatives of the four groups were tested against mutants in all other groups of the KOS series (Table 4). Only one, ts508, failed to complement a mutant in a previously defined group (tsBZb). Therefore, three of the four groups were new. Mutants derived from mutagenesis of fragments were designated as follows: ts508 is tsB28h; ts701, tsT34h; ts797, tsT36h, ts704, tsU35h; and ts953, tsV37h. Complementation data were thus consistent with the prediction that new mutants should represent either unmapped groups or that they should belong to group B. Complement&ion Tests between HA-Induced Mutants and Mutants in the Standard Set In order to determine whether mutants in groups Q, R, S, T, U, and V of HSV-1 strain KOS were in the same or different groups defined by the standard set of HSV1 ts mutants @chaffer et al., 1978), complementation tests were conducted with representatives of groups l.-16 through l.-23. Groups 1. - 1 through 1. - 15 in the standard set contain KOS ts mutants with which HAinduced mutants had already been tested (see above). As seen in Table 5, complementation was demonstrated between mutants in all pairs. Therefore, KOS groups Q, R, S, T, U, and V constitute new HSV-1 complementation groups (l.-24 through l.-29) bringing the total number of groups defined by ts mutants to 29. 4

COMPLEMENTATIONBETWEENHA-INDUCEDMUTANTSANDMUTANTSINE PREVIOUSLYIDENTIFIED COMPLEMENTATIONGROUPS Complementation ts mutants 508 701 704 797 953

index”

Al

B2

C7

D9

E6

F18

G3

HlO

Ill

J12

Kl3

L14

Ml9

N20

022

34 3700 18 15

0.9 160 48 280 95

420 16 250 11

57 5 34 15 25

190 4 32 26 24

81 22 4 52 21

880 110 6 58 41

704 71 6.2 150

3000 78 2.444 1800

46 200 4 340 110

240 3 420 13

2.6 25 20 2.6

230 4 32 37

220 9 8 27

160 9--7 32

(1See footnote b See footnote

to Table 1 b. Table 2.

P23O

yZ6

68

:,

29 90 29

11 12 2X

H30 160 9.2 ox 6

S91 ZPO 9.5 170 76

176

CHU ET AL. TABLE

COMPLEMENTATION

BETWEEN

HA-INDUCED

5

ts MUTANTS

AND

ts MUTANTS

Complementation

ts mutant tsQ26h tsR30h tsS3lh tsT36h tsU35h tsV37h

IN THE HSV-1

STANDARD

SET

indexa

Ab (l-16)

478 (I-17)

7 (l-18)

Bl (1-19)

B7 (l-20)

LB4 (l-21)

LB5 (l-22)

140 64 57 43 13 20

30 56 11 21 5.7 26

6.2 38 10 5.6 28 18

380 15 500 5500 400 360

290 5.6 47 530 63 28

100 43 7.5 220 19 13

200 5.7 6.1 460 18 16

D10 (l-23) 210 19 16 570 76 76

a See footnote to Table 1. b Mutants and complementation groups in the standard set are described by Schaffer et al. (1978). Numbers in parentheses represent complementation groups in the standard set.

Mapping

of HA-Induced

Mutants

In an effort to determine the location of HA-induced mutants on the viral genome, two approaches were used. The first, complementation analysis, can give only indirect evidence concerning the location of a mutant, while the second method, marker rescue, gives the location directly. Because mutants in six complementation groups had been mapped previously (Parris et al., manuscript in preparation), HA-

11736h Hid

induced mutants which failed to complement (or recombine with) these mutants, and thus belong to the same complementation group, should lie at or near the location of the corresponding, previously mapped mutant. Figure 5 illustrates the map locations of mutants in three groups of the standard set: tsF18g, tsJ12g, and tsB2lu. tsQ26h and tsR30h represent new cistrons which cannot be located indirectly by the results of complementation tests with previously mapped mutants. Nevertheless,

H

0

I

1

4

K

t

0

c

s

M

N

0

a0

0.0

a0

a,

0.0

2.0

0.0

a0

00

0.0

0.0

0.0

0.0

0.0

0.0

0.0

a0

0.0

0.0

0.0

0.0

0.0

a0

0.0

0.0

II

Fl

,,g..,

111 ls”17h

0.0

0.0

0.0

0.5

00

1.5

010

4. Marker rescue oftsT36h and tsV37h. Physical maps ofHind andXba1 HSV-1 (KOS) DNA fragments are presented (Skare and Summers, 1977). The heavy lines on the maps indicate the positions of mutagenizedEcoR1 A and B fragments. The table gives the efficiency of rescue of each mutant DNA by individual wild-type DNA fragments. Total yields of infectious virus from mixed infections with ts mutant DNA and wild-type viral DNA fragments ranged from 10” to 106. Values are expressed as (PFU 39YPFU 34”) x 100. FIG.

MUTAGENESIS

177

OF HSV DNA FRAGMENTS

*

,+,

,&

MUTAGtW1ZED FRAGMRNIS

FIG. 5. Physical map locations of HA-induced ts mutants. The positions of tsF23g, tsJ12g, and tsB2lu are from Parris et al. (in preparation). The locations of tsB27h, tsB28h, tsB32h, tsF29h, and tsJ33h were determined by complementation analysis; those of tsT36h and tsV37h were determined by marker rescue.

recombination frequencies between these two mutants and tsD9b were low (less than O.l%, data not shown). Although recombination by two-factor crosses is not a reliable method for locating mutants on the physical map of the HSV-1 genome because recombination distances are not accurately reflected on the physical map (Parris et aE., manuscript in preparation), these data indicate that tsQ26h and tsR30h lie near tsD9 on the physical map. tsD9b is located between coordinates 0.339 and 0.453. In an attempt to demonstrate that the mutants derived from mutagenized fragments were in fact located in the region of the intact genome from which fragments A and B were derived, marker rescue experiments were performed. Here, wildtype virus fragments were derived from Hind111 or XbaI cleaved HSV-1 DNA and used to rescue ts lesions. tsT36h and tsV37h were both rescued efficiently by fragments containing sequences in the EcoRI A fragment (Figure 4). HindIII-K and XbaI-E fragments rescued tsT36h with efficiencies of 2.0 and 2.5%, respectively. A low frequency of rescue was observed with HindIIIJ (0.8%). However, because this fragment migrates in gels immediately behind the K fragment, the observed rescue was probably due to contamination of HindIII-J with HindIII-K. This finding becomes more likely if one considers that rescue was observed only with fragment E of the XbaI fragments. As HindIII-K is contained

within XbaI-E, the limits within which tsT36h lies are 0.530-0.594 (Fig. 5). Similar results were observed with tsV37h (Fig. 4). HindIII-K and XbuI-E rescued with efficiencies of 1.5 and 6.2%, respectively. Again a low frequency of rescue was observed with HindIII-J (0.5%); however, because the fragments were from the same preparation as used with tsT36h, it is probable that this also was due to fragment contamination. With this mutant low levels of rescue were also seen with XbuI-C and F, but because these fragments are difficult to separate from X&I-E and because the E fragment rescued with sixfold greater efficiency, it was felt that this rescue was probably due to contamination of XbaI-C and F with E. Thus, as in the case oftsT36h, tsV37h lies between coordinates 0.530 and 0.594, and within the limits of EcoRI fragment A (Fig. 5). Taken together, the aforementioned data demonstrate that mutations have been introduced into HSV-1 DNA sequences represented by EcoRI fragments A and B. Viral

DNA Phenotypes Mutants

qf HA-Induced

In order to compare the DNA phenotypes of HA-induced mutants with those of previously described mutants, viral DNA phenotypes were determined. The viral DNA phenotypes of mutants are based upon the ability of mutant-infected cells

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to synthesize viral DNA at 39” compared with cells infected with the wildtype virus at this temperature. Mutants which induce the synthesis of more than 20% of the wild-type level of viral DNA are considered to be DNA+; mutants which induce the synthesis of 20% or less are considered to be DNA’. At 34” cells infected with all HA-induced mutants synthesized more than 50% of the wild-type level of HSV DNA (Table 6). At 39”, mutants shown to be DNA+ included tsF29h, J33h, R30h, S39h, T34h, U35h, and V37h; DNA’ mutants included tsB2’7h, Q26h, S38h, and T36h; and DNA- mutants included tsB32h, B28h, and S31h. Based on the data presented in Table 6, some heterogeneity in the viral DNA phenotype exists among mutants belonging to the same complementation group. tsB27h, a DNA’ TABLE

6

VIRAL DNA PHENOTYPES OF WILD-TYPE VIRUS AND HA-INDUCED ts MUTANTS AT 34 AND 39”” Viral DNA Source

Virus Wild-type virus

34”

100 loo(+)*

HA-treated virions

tsB27(277)C

72

HA-treated HSV DNA (11 min)

tsF29(201)

77 86

HA-treated HSV DNA (13 min)

tsB32(401) tsJ33(449) ts R30(484) f&38(487)

tsQ26(372)

tsS39(488)

t&31(494) HA-treated HSV DNA fragments

tsB28(508) tsT34(‘701) tsU35(704)

tsT36(797) LTV37(953)

39

88 122 59 61 58 67

5.4(k) 43(+) 12(k) O(-1

26(+) 33(+) 6.5(k) 29(+) w-1

37 0(--j 75 126(+) 104 113(+) 71 12(k) 96 108(+)

B Results are expressed as the percentage of viral DNA in wild-type virus-infected cells. Values represent the average of two separate experiments. b +, 2, and - are viral DNA phenotypes; DNA+ = >20%, DNA’ = ~20%, DNA- = none detectable. c Numbers in parentheses are original mutant designations.

mutant, belongs to group B which also contains the DNA- mutants tsBBb, B21u, B28h, and B32h. The DNA’ phenotype of tsB27h may result from the unusually high level of leak exhibited by this mutant. Similarly tsS38h, S39h, and S31h synthesized varying amounts of DNA at 39”. While the reason for the discrepancy remains to be determined, the possibility of multiple mutations has not been ruled out. The DNA+ phenotypes of tsF39h and tsJ33h are consistent with those of other members of the F and J groups (Schaffer et al., 1973). DISCUSSION

This report describes a method for the introduction of mutations into preselected regions of the HSV-1 genome. Prerequisites for the utilization of this procedure include methods for the isolation and purification of restriction enzyme-generated DNA fragments, the availability of physical maps of these fragments, a mutagen which acts on nonreplicating DNA and the demonstrated ability of fragments to recombine with the intact genome following transfection. Because of the simplicity of the procedure, mutants in small, specialized DNA sequences of interest may be isolated rapidly. Initially, HA was chosen as the mutagen because of its specific interaction with cytosine residues. Because most of the HSV-1 mutants which have been isolated previously were derived using thymine-specific mutagens @chaffer et al., 1978) and because HSV-1 has a G:C content of 67% (Kieff et al., 1971; Graham et al., 1972), a cytosine-specific mutagen seemed a good choice for the identification of new cistrons. As pointed out earlier, during mutagenesis of virions HA possesses nonmutagenic, virus-inactivating properties. Although we attempted to use conditions which had been shown to limit the inactivating properties of HA in phage systems, the low rate of mutant recovery following mutagenesis of virions (l/458 as opposed to 81459 for intact DNA) probably reflects the inactivating property of HA. Thus, HA was not an efficient mutagen of virions under the conditions we employed for mutagenesis.

MUTAGENESIS

OF

HSV

HA mutagenesis of purified DNA does not suffer from this complication, however. Because HA acts almost exclusively on cytosine residues in single-stranded DNA, it was felt that it should be possible to select conditions which would limit the number of hits on a molecule and permit the isolation of single mutants. Therefore, a lower concentration of HA (0.05 M) was used in an effort to reduce the total number of mutations per molecule. As demonstrated, this concentration of HA yielded a good correlation between the level of inactivation of the DNA as a function of the duration of treatment and the proportion of mutants isolated at a given time. Thus, while other investigators recommend the use of higher concentrations of HA (Budowsky, 1976), we have found 0.05 M to be satisfactory. With regard to the number of mutations per DNA molecule, it is significant that no double ts mutants were detected in tests with mutants representing 23 complementation groups. Because HA is mutagenic primarily on single-stranded DNA (Freese and Strack, 1962), mutagenesis was conducted under partially denaturing conditions at 70”. Total denaturation results in a loss of greater than 90% of infectivity; therefore, it becomes counterproductive to denature intact DNA to this extent. At 70” in 0.05 x SSC and 0.05 M HA, only 5-10% of HSV-1 DNA should have been denatured. Thus, theoretically, mutagenesis was limited primarily to cytosine residues in A:T rich regions of the genome. At present only three of the six groups of mutants induced by HA mutagenesis of intact DNA have been mapped (Fig. 5). Two of these mutants (tsF29h and tsJ33h) have been shown to map in A:T rich regions of the genome (Fig. 5, Hayward et al., 1975). Additionally, there is evidence from recombination analysis that two other groups (tsQ26h and tsR30h) also lie in A:T rich regions but direct mapping of these groups has not yet been completed. tsB32h lies in the G:C rich repeated sequences bounding the Us region of the genome (Parris et al., manuscript in preparation); the high G:C content of this region may have presented more target cytosine residues: Local “breathing” in the molecule could account for briefly dena-

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FRAGMENTS

179

tured regions here. Furthermore, the B gene is diploid (Knipe et al., 19’78), thus presenting a greater target size. The presence of nicks and gaps in the DNA may also permit cytosine-specific mutagenesis at sites which would not normally be denatured. Thus, the peculiar properties of HSV DNA may account not only for the location of mutations in non-A:T rich regions, but also for the efficiency of HA mutagenesis. The use of mutagenized fragments to introduce mutations into preselected regions of the HSV genome has been explored. The procedure described here offers the advantage that any small region of the genome can be selectively mutagenized. In this regard, it should be noted that we do not know the lower size limit of the DNA fragments required for marker rescue. To date, the smallest defined fragment shown to rescue in our hands is 1.7 x 10” (Parris et al., manuscript in preparation). We have, however, demonstrated rescue with randomly sheared DNA (300-500 bp) which rescued with lower efficiency. Should rescue prove successful with smaller, defined fragments, it may be possible to introduce a number of mutations into the same gene permitting fine structure genetic analysis. Previous reports have dealt with methods of site-specific mutagenesis which permit alteration of specific bases near restriction enzyme sites in an intact viral genome or bacterial plasmid (Weissman et al., 1977; Shortle and Nathans, 1978; Heffron et al., 1978). While these techniques are of use for fine structure analysis of genes whose precise map coordinates are known and for which detailed physical maps are available, they are limited in that they require such detailed physical mapping data as well as large quantities of the DNA of interest. The method described here allows the mutation of a gene which may or may not have been identified, does not require precise physical mapping data, is not dependent upon circularization of the molecule (Shortle and Nathans, 1978; Heffron et al., 1978), and requires only small amounts of DNA. For genetically complex viruses such as HSV, the use of random mutagenesis is a tedious process at best. Because restriction enzyme maps of HSV DNA of sufficient

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detail are not yet available for use in the site-specific mutagenesis procedures just described, mutagenesis of isolated viral DNA fragments was attempted. As demonstrated in the present report, mutagenesis of fragments has resulted in the rapid identification of three new cistrons in DNA sequences which were previously mutationally silent. It can be anticipated that with the use of more, and increasingly smaller fragments, a large number of viral genes should be identified quickly. Additionally, regions of the genome which are of special interest structurally or functionally (Morse et al., 1978) can be enriched for mutations. Finally, other animal virus systems such as poxviruses and adenoviruses which have large, complex genomes should lend themselves to analysis by this method as readily as HSV. ACKNOWLEDGMENTS We thank V. C. Carter, G. P. Makari, P. A. Temple, and L. B. Sandner for excellent technical assistance. This investigation was conducted in part in the Department of Virology, Baylor College of Medicine, Houston, Texas, supported by Public Health Service Research Contract CP 53,526 within the Virus Cancer Program of the National Cancer Institute and Research Grant CA 10,893 from the National Cancer Institute. The work conducted in Boston was supported by Research Grant CA 20,260 and Program Project Grant CA 21,082 from the National Cancer Institute. D.S.P. was the recipient of Postdoctoral Fellowship CA 5,465 from the National Institutes of Health. REFERENCES ARON, G. M., PURIFOY, D. J. M., and SCHAFFER, P. A. (1975). DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants. .Z. Viral. 16, 498-507. BUDOWSKY, E. I. (1976). The mechanism of mutagenic action of hydroxylamines. In “Progress in Nucleic Acid Research and Molecular Biology,” (W. E. Cohn, ed.), Vol. 16, pp. 125-187. Academic Press, New York. DELIUS, J., and CLEMENT% J. B. (1976). A partial denaturation map of herpes simplex virus type 1: Evidence for inversions of the two unique DNA regions. J. Gen. Viral. 33, 125-133. HEFFRON, F., So, M., and MCCARTHY, B. J. (1978). In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites. Proc. Nat. Acad. Sci. USA 75, 6012-6016. FARBER, F. E. (1976). Comparison of DNA facilitators

in the uptake and intracellular fate of infectious herpes simplex virus type 2 DNA. Rio&em. Biophys. Acta 454, 410-418. FREESE, E., and STRACK, H. B. (1962). Induction of mutations in transforming DNA by hydroxylamine. Proc. Nat. Acad. Sci. USA 48, 1796-1803. FREESE, E., BAUTZ-FREESE, E., and BAUTZ, E. (1961). Hydroxylamine as a mutagenic and inactivating agent. J. Mol. Biol. 3, 133-143. GRAHAM, B. L., LUDWIG, H., BRONSON, D. L., BENYESH-MELNICK, M., and BISWAL, N. (1972). Physicochemical properties of the DNA of herpes viruses. Biochem. Biophys. Acta 259, 13-22. HAYWARD, G., JACOB, R. J., WADSWORTH, S. C., and ROIZMAN, B. (1975). Anatomy of herpes simplex virus DNA: Evidence for four populations of molecules that differ in the relative orientations of their long and short components. Proc. Nat. Acad. Sci. USA 72, 4243-4247. KIEFF, E. D., BACHENHEIMER, S. L., and ROIZMAN, B. (1971). Size, composition, and structure of the deoxyribonucleic acid of herpes simplex virus subtypes 1 and 2. J. Viral. 8, 125-132. KNIPE, D. M., RUYECHEN, W. T., ROIZMAN, B., and HALLIBURTON, I. W. (1978). Molecular genetics of herpes simplex virus: Demonstration of regions of obligatory and non-obligatory identity within diploid regions of the genome by sequence replacement and insertion. Proc. Nat. Acad. Sci. USA 75,3896-3900. MARSDEN, H. S., STOW, N. D., PRESTON, V. G., TIMBURY, M. C., and WILKIE, N. M. (1978). Physical mapping of herpes simplex virus-induced polypeptides. J. Viral. 28, 624-642. MORSE, L. S., PEREIRA, L., ROIZMAN, B., and SCHAFFER, P. A. (1978). Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV 1 x 2 recombinants. J. Viral. 26, 389-410. ROBB, J. A., and MARTIN, R. G. (1970). Genetic analysis of simian virus 40. I. Description of microtitration and replica-plating techniques for virus. Virology 41, 751-760. SCHAFFER, P. A., ARON, G. M., BISWAL, N., and BENYESH-MELNICK, M. (1973). Temperaturesensitive mutants of herpes simplex type 1: Isolation, complementation, and partial characterization. Virology

52, 57-71.

SCHAFFER, P. A., TEVETHIA, M. J., and BENYESHMELNICK, M. (1974). Recombination between temperature-sensitive mutants of herpes simplex virus type 1. Virology 58, 219-228. SCHAFFER, P. A., CARTER, V. C., and TIMBURY, M. C. (1978). Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus type 1 and 2. J. Viral. 27, 490-504. SHELDRICK, P., and BERTHELOT, N. (1974). Inverted repetitions in the chromosome of herpes simplex virus. Cold Spring Harbor Symp. Quant. Biol. 39, 667-681.

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SHORTLE, D., and NATHANS, D. (1978). Local mutagenesis: A method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc. Nat. Ad. Sci. USA 75, 21’70-21’74.

SKARE, J. and SUMMERS, W. C. (1977). Structure and function of herpes simplex virus genome. II. EcoRI, XbaI, and Hind111 endonuclease cleavage sites in herpes simplex virus type 1 DNA. Virology 76, 581-595.

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WADSWORTH, S., JACOB, R. J., and ROIZMAN, B. (19’75). Anatomy of herpes simplex virus DNA. II. Size, composition, and arrangement of inverted terminal repetitions. J. Viral. 15, 1487-1497. WEISMANN, G., TONIGUCHI, T., DOMINGO, E., SABO, D., and FLAVELL, R. A. (1977). Site directed mutagenesis as a tool in genetics. In Genetic Manipulation as It Affects the Cancer Problem (J. Schultz and Z. Brada, eds.), pp. 11-36. Academic Press, New York.