JOURNAL OF VIROLOGY, June 1978, p. 547-553 0022-538X/78/0026-0547$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 26, No. 3 Printed in U.S.A.

Analysis of dCMP Deaminase and CDP Reductase Levels in Hamster Cells Infected by Herpes Simplex Virus YVES LANGELIER,t MARGUERITE DECHAMPS, AND GERARD BUTTIN*

Unite de Genetique Cellulaire, Institut de Recherches en Biologie Moleculaire, Centre National de la Recherche Scientifique, 75221 Paris, Cedex 05, France Received for publication 28 December 1977

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster ([A1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity. Herpes simplex virus (HSV) induces several enzymes related to DNA synthesis. There is strong biochemical, immunological, and genetic evidence that, among these enzymatic activities, at least deoxypyrimidine kinase, behaving both as a thymidine kinase (TK) and as a deoxycytidine kinase (10, 11), and a DNA polymerase are virally coded products (4, 12, 14-16, 27, 34, 35, 37; P. A. J. Perera and J. M. Morrison, Biochem. J. 117:21p-22p, 1970). The demonstration by Munyon et al. (23) that the deoxypyrimidine kinase gene could be transferred to TK- mouse cells, thus permitting these cells to survive in HAT medium (20), opened the way for introducing external genetic information into an eucaryotic cell line. Interest in this new transformation system was enhanced when Davidson et al. (7) found that in some instances the activity of the viral enzyme was shut off upon cultivation of the transformed cells in nonselective medium, although the HSV deoxypyrimidine kinase gene itself was still retained. The possibility that certain HSV-specified products might regulate the expression of the viral deoxypyrimidine kinase in transformed cells was suggested by the finding that superinfection of these cells by HSV TK- mutants enhanced the activity of the viral TK (13, 18, 19). Understanding the mechanisms that regulate

the expression of a latent viral gene in transformed cells is of obvious importance; so far, the deoxypyrimidine kinase system is the only one that has been exploited at the biochemical level for such a purpose. It is therefore very desirable to develop additional transformation systems so that the regulation of the expression of more than one viral gene can be studied in the same cell. Other enzymes involved in the synthesis of nucleotide precursors have been reported to be induced upon infection by HSV: a dCMP deaminase (32), a deoxycytidine deaminase (2), and a CDP reductase (5). The availability of dCMP deaminase-deficient Chinese hamster cells (30) and the establishment of a selection system for this enzymatic activity in our laboratory (B. Robert de Saint Vincent, unpublished data) led us to test the practicality of the HSVinduced dCMP deaminase as a second marker for genetic transformation. For such a purpose, the inducible CPD reductase should also be a good candidate, because of the reported insensitivity of the induced enzyme to dTTP (5), a property that could serve as a basis for selecting stable acquisition of this activity by mammalian cells. As a first step in establishing a selection system for the dCMP deaminase marker, we wished to determine, with the help of mutant cells dePresent address: Institut du Cancer de Montreal, Mon- void of the enzymatic activity, whether the ret ported induction of dCMP deaminase after HSV treal, Quebec, Canada H2L 4M1. 547

548

LANGELIER, DECHAMPS, AND BUTTIN

J. VIROL.

infection represented the synthesis of a new enzyme rather than the stimulation of a cellular isozyme. Confirmation of the induction of CDP reductase activity was also desirable. We found, as will be documented by the results to be presented, that no significant induction of dCMP deaminase occurred either in dCMP de deaminase occurred either in mutant dCMP deaminase-negative cells or in quiescent BHK21/C13 cells after infection by HSV type 1 (HSV-1) or HSV-2, under conditions in which both TK and CDP reductase were strongly induced. MATERIALS AND METHODS

mM dithiothreitol, containing either 5 x 10' M dCTP

Cell lines. CCL39 is a line of female Chinese hamster lung fibroblasts supplied by the American Type Culture Collection. lAl, its dCMP deaminase-negative derivative, was isolated for resistance to 0.5 jig of 1-,BD-arabinofuranosylcytosine per ml by B. Robert de Saint-Vincent, utilizing a one-step procedure as described previously (8). The BHK-21/C13 cell line was obtained from the Pasteur Institute. All cell cultures were normally grown at 37°C in a modified Eagle medium containing the nonessential amino acids and enriched by doubling the normal amounts of glucose, vitamins, and amino acids (essential and nonessential). This Eagle reinforced (ER) medium was normally supplemented with 10% fetal calf serum (ER1o). The absence of mycoplasm was periodically tested by microbiological and staining assays kindly performed for us by the Service des Virus at the Pasteur Institute. HSV-1 strain F was obtained from P. Sheldrick, and HSV-2 strain HG-52 was obtained from J. SubakSharpe. The two strains of viruses were passaged at low multiplicities in cultures of BHK-21/C13 cells grown in ER2. Viral stocks of high titers were prepared by the procedure of Roizman and Spear (31). Plaque titrations were performed in 60-mm petri dishes, essentially by the method of Wentworth and French (38). Induction of enzymatic activities in virus-infected cells. Glass roller bottles were each seeded with 2 x 107 cells in ER1o; confluent cultures were obtained after 3 days of incubation at 37°C. For the production of quiescent cultures, plastic roller bottles (Corning Glass Works, Corning, N.Y.) were used to prevent cell detachment; after 24 h of incubation in ER1o, the monolayers were washed with ER and further incubated in the presence of ER0.5 for 4 to 5 days at 37°C. The cultures were infected at an input multiplicity of 20 to 50 PFU/cell, and the virus was allowed to adsorb at 37°C for 1 h. The medium containing unattached viruses was then replaced by ER2 (or ERo05 for quiescent cultures), and the incubation was continued at 34°C as indicated. Preparation of enzyme extracts. After two washes of the monolayers with 20 ml of 0.1 M phosphate buffer (pH 7.5) containing 0.1 M KCl, the cells were detached from the substratum with i0' M EDTA in the same buffer, separated into portions for the different enzymatic determinations, and collected by low-speed centrifugation. They were washed in phosphate buffer without EDTA and suspended in a small volume of 0.05 M Tris-hydrochloride (pH 7.8)-i

supernatant fluid was collected. TK assay. TK assays were performed by the

mutant

for the determination of dCMP deaminase and CDP activities or 0.1 mM thymidine for the determination of TK activity. The cell suspensions were frozen at and could be stored for months without loss of

-70°C

enzymatic activities. For dCMP deaminase and CDP reductase assays, the thawed cells were disrupted by

homogenization (80 strokes) in a Dounce homogenizer. In some experiments, homogenization was carried out with Brij 35 (0.5% wt/vol), followed immediately by the addition of ethyleneglycol to a final concentration of 20% (vol/vol). For the TK assay, the cells were sonically treated for 2 min at 8/10 of maximal power of a Siduse sonic oscillator. The suspension was then centrifuged at 10,000 x g for 10 min at 4°C, and the method of Caboche (1). The incubation mixture contained in a final volume of 100 ,l: 50 mM Tris-hydrochloride (pH 8.0), 2.5 mM MgCl2, 2.5 mM ATP, 20 /M thymidine plus 0.2 MCi of [methyl-'H]thymidine, and from 5 to 20 Ml of enzyme extract. The mixture was incubated at 37°C. At different intervals, 20-Ml samples were spotted onto Whatman DE81 paper squares (2 cm2). The reaction was linear for up to 30 min. The squares were washed twice (30 min) in 2 x 10- M ammonium formate, once in distilled water, and finally once in absolute ethanol. After being dried, they were placed in scintillation vials containing a 2,5-diphen-

yloxazole-1,4-bis- (5-phenyloxazolyl)benzene-toluene mixture, and the radioactivity was determined in an Intertechnique SL40 liquid scintillation spectrometer. dCMP deaminase assay. The dCMP deaminase was measured by a method developed from that described by Maley and Maley (21). The incubation mixture contained in a total volume of 40 Il: 25 mM Tris-hydrochloride (pH 8.0), 1 mM MgCl2, 20 yM dCTP, 1 mM dCMP plus [5-3H]dCMP (7 x 105 cpm counted on polyethyleneimine-cellulose), and enzyme extract (100 to 400 Mig of protein). The mixture was incubated at 37°C for 15 min, and the reaction was stopped by immersing the tubes in boiling water for 2 min. The precipitate was removed by centrifugation at 10,000 x g for 10 min, and 10-Ml portions of the supernatant were spotted onto a polyethyleneiminecellulose sheet together with nonradioactive dUMP and dCMP, which served as markers. The product was separated from the substrate by unidimensional migration first in 1 N acetic acid for 15 min and then in 0.3 M LiCl for 40 min. After localization by examination in UV light, the appropriate spots were cut out and the radioactivity was measured as described for the TK assay. CDP reductase assay. To remove deoxynucleotides, the cell extract was passed through a small column of cation-exchange resin (AG1-X8, Bio-Rad Laboratories, Richmond, Calif.) previously equilibrated with 0.05 M Tris-hydrochloride (pH 7.8) containing 1 mM dithiothreitol (25). The standard reaction mixture contained in a total volume of 185 Ml: 5.4 mM Tris-hydrochloride (pH 7.0), 0.054 mM FeCl:, 4.3 mM NaF, 6.5 mM magnesium acetate, 2.7 mM ATP, 5.4 mM dithioerythritol, 0.054 mM CDP plus 105 cpm of [3H]CDP, and enzyme extract (200 to 1,000 Mig of protein). After incubation for 60 min at 37°C, the

activity

VOL. 26, 1978

ENZYME INDUCTION IN HSV-INFECTED CELLS

reaction was stopped by the addition of 180 ,u of 2 N perchloric acid, and the precipitate was removed by centrifugation. Nucleotides in the supernatant were converted to their monophosphate forms by boiling them at 100°C for 10 min; dCMP was separated from CMP by column chromatography on AG 50W-X8 (Bio-Rad Laboratories) by the method of Reichard

549

7

CCI 39

IAI

(29).

Chemicals and radiochemicals. CMP, CDP, CTP, dUMP, ATP, and dATP were obtained from Sigma Chemical Co., St. Louis, Mo.; dCMP and dTTP were from P. L. Biochemicals Inc., Milwaukee, Wis.; [methyl-3H]thymidine (45 Ci/mmol) was from CEA, Saclay, France; [5-3H]dCMP (15 Ci/mmol) was from New England Nuclear Corp., Frankfurt, Germany; and [5-3H]CDP (13 Ci/mmol) was from Amersham/Searle, Arlington Heights, Ill. [5-3H]dCMP and [5-3H]CDP were purified by ionic-exchange chromatography. Polygram CEL 300 PEI/UV254 plastic sheets for thinlayer chromatography were purchased from Macherey-Nagel and Co., Diiren, Germany.

RESULTS HSV replication in CCL39 cells. CCL39 cells, as previously reported by Rapp and Hsu (28), are less susceptible to HSV infection than

/ X X \ \/

2 X

5

i

6 2 it

Time post infection (h)

are rabbit kidney cells or BHK-21/C13 cells. Stocks of HSV-1 strain F and HSV-2 strain HGFIG. 1. Synthesis HSV-1 strain in infectiousmonolayers strain cells. of wereFinH, _CCL39 Confluent 52, which HSV*1 cells fected atoranlAIinput gave high titers on BHK-21/C13 multiplicity of 5 PFU/cell. At the (4 x 10 and 4 x 107 PFU/ml, respectively), gave indicated times, the viruses were extracted from triplower titers on CCL39 cells (8 x 105 and 3 x 105 licate cultures by three cycles offreezing and thawing PFU/ml, respectively). The host-dependent dif- and titrated as described in the text. ferences raised the question as to whether the two strains of viruses have normal growth cycles the activities of some other enzymes known to on CCL39 cells. Representative growth curves be virally induced were indeed expressed in this are shown for HSV-1 (Fig. 1) and HSV-2 (Fig. 2) cell-virus system. In the experiments shown in with cells of CCL39 or of its lAl derivative as Table 2, TK and CDP reductase activities were host. There was no difference between the wild- measured in addition to that of dCMP deamitype and mutant cells in their capacity to sup- nase. The results clearly showed that although port viral replication, but for both cell lines the infection of lAl cells with HSV-1 or HSV-2 did growth cycle was slower and the yield of infec- not induce detectable dCMP deaminase activity, tious virions was considerably reduced as com- strong induction of TK and CDP reductase acpared with BHK infection. tivities did occur. TK activity was enhanced dCMP deaminase activity in mutant and about 25-fold upon HSV-2 infection and 10-fold wild-type cells. Several dCMP deaminase-neg- upon HSV-1 infection, whereas CDP reductase ative clones were identified among survivors of activity, which was measured only in HSV-1mutagenized CCL39 cells in a medium contain- infected cells, was raised 8-fold. ing 0.5 ,ug of 1-fl-D-arabinofuranusylcytosine per As reported by Cohen (5) for the CDP reducml (30). As shown in Table 1, the dCMP deam- tase activity induced in HeLa cells upon infecinase activity was below detection in extracts tion by HSV-1, the CDP reductase activity inprepared from one of these mutants (IA1) in duced in Chinese hamster cells was also found exponential growth. A mixture of mutant and refractory to inhibition by dTTP. This is shown wild-type cell extracts, in equal amounts, gave in Fig. 3, where the sensitivity to dTTP of CDP an intermediary specific activity suggesting the reductase activity in extracts of HSV-1-infected absence of an inhibitor in the mutant extract. confluent lAl cells and in uninfected exponendCMP deaminase, TK, and CDP reduc- tially growing lAl cells was compared. At 0.05 tase activities in HSV-infected [Al cells. mM, dTTP inhibited the host cell enzyme by Since in preliminary assays we did not detect 50%; at 0.2 mM, the inhibition reached 90%. On dCMP deaminase activity in HSV-1- or HSV-2- the other hand, inhibition in extracts of infected infected lAl cells, it was important to verify that cells was never greater than 30%, even at 1.0

LANGELIER,

550

DIWCHAMPS,

J. VIROL.

AND BUTTIN

7-

ca 39

IAI

S

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I, . 24 & 24 16 24 32-~ 24 Time post infection (h) FIG. 2. Synthesis of infectious HSV-2 strain HG52 in CCL39 or LAJ cells. Infection and titration were done as described in the legend to Fig. 1. 5

a

18 16

TABLE 1. Activity of dCMP deaminase in wild-type

and mutant cells Cell line

tracts

(nmol 30 min-' mg-')

CCL39 (wild type) lAl CCL39 + lAl (1:1)

53