Factors Governing Expression of the Herpes Simplex Virus Gene for Thymidine Kinase in Clonal Derivatives of

Transcription

1 JOURNAL OF VIROLOGY, Jan. 1981, p X/81/ $2./ Vol. 37, No. 1 Factors Governing Expression of the Herpes Simplex Virus Gene for Thymidine Kinase in Clonal Derivatives of Transformed Mouse L Cells RALPH BUTTYANt AND PATRICIA G. SPEAR* Department ofmicrobiology, University of Chicago, Chicago, Illinois 6637 The cells used in this study are sublines of a transformed mouse L cell line (designated H2) that carries the herpes simplex virus (HSV) gene for thymidine kinase (tk) as well as other viral genetic information acquired after exposure of the parental Ltk- cells to UV-irradiated HSV type 1. These sublines of the H2 cell line were isolated by cloning under nonselective conditions and were shown to express widely different levels of viral tk. Selective media were used to isolate phenotypically tk- and tk+ variants in sequence from one of the clonal derivatives. As previously reported, superinfection of the tk+ cell lines with tk- HSV type 1 resulted in enhancement of tk activity. A new finding was that viral tk activity could be induced by superinfection in at least 3% of cells from the phenotypically tk- sublines, indicating that a functional viral tk gene was retained in a significant proportion of the cells. Experiments were designed to test for the presence of regulatory factors that could influence tk expression in the nonsuperinfected sublines of H2. Absence of freely diffusible regulatory factors was indicated by the finding that the fusion of phenotypically tk- and tk+ cells and untransformed cells in appropriate combinations did not affect the levels of tk detected. Moreover, there was no evidence for the presence in phenotypically tk+ transformed cells of HSV-specific regulatory factors that could influence expression of tk from a superinfecting viral genome. Phenotypically tk+ sublines of H2 were found to differ from the phenotypically tk- sublines and from untransformed cells in that the tk+ cells synthesized viral proteins earlier and produced greater yields of infectious HSV progeny after superinfection with wild-type tk+ virus. We can conclude that the absence of tk expression in the tk- H2 sublines cannot be accounted for by rearrangements or loss of DNA sequences encoding the enzyme itself or of sequences necessary for induction of the gene by superinfecting HSV. Moreover, it appears that the expression of tk in the tk+ H2 sublines correlates with the presence of some factor that can enhance (or the absence of some factor that can depress) HSV replication and gene expression. Thymidine kinase (tk)-deficient mouse cells can be transformed to the tk+ phenotype by infection with UV light-inactivated herpes simplex virus (HSV) (5, 13, 3, 34) or by introduction of HSV DNA fragments (27, 27a, 31, 41). These tk+ transformants (hereafter referred to as LVtk+ cells) synthesize tk of viral origin (5, 13, 29, 39, 41), contain portions of the viral genome (6, 22, 24, 31, 38; J. M. Leiden, Ph.D. thesis, University of Chicago, Chicago, 111., 1979) and express virus-specific antigens (2, 3, 2). Davidson et al. (5) reported that phenotypically tkcells could be isolated from certain LVtk+ cell lines at a relatively high frequency. They also showed that some of the revertant cells retained the viral tk gene and that the gene could be t Present address: Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, NJ 711. reactivated in at least a fraction of the cells, because phenotypically tk+ clones were isolated from the tk- revertants and found to express viral tk. Thus, some regulatory systems in certain transformed cell lines can modulate expression of the resident viral tk gene. During productive infections with HSV, expression of tk from the viral genome appears to be regulated by other viral products. Experiments done with metabolic inhibitors (8) and with temperature-sensitive mutants of HSV-1 (32) demonstrated that production of functional tk mrna requires the prior synthesis of functional early viral proteins and transcription in the presence of these proteins. Viral DNA synthesis is not required for tk synthesis, and, in fact, inhibition of DNA synthesis results in overproduction of tk (8). HSV tk belongs, therefore, to the,b class of viral proteins, whose synthesis 459

2 46 BUTTYAN AND SPEAR is induced by the prior synthesis of viral a proteins and is repressed by the synthesis of late products (11, 12, 35, 36). It was previously shown that expression of the viral tk gene resident in transformed mouse L cell lines can be influenced by products of superinfecting HSV. Specifically, the levels of tk activity detectable after infection of LVtk+ cells with tk- HSV were significantly enhanced over those in mock-infected control cells (17, 23, 25), and higher levels of tk activity were attained in the absence of viral DNA synthesis (23). The results of experiments done with metabolic inhibitors (23) and with a temperature-sensitive mutant of HSV (19) indicated that expression of an early (a) protein from the input viral genome was required for the enhancement of tk activity and that expression of late viral products blocked further increases in levels of tk. In this paper, we report investigations of factors which could regulate expression of the HSV tk gene present in transformed mouse L cells, with some emphasis on the possibility that viral regulatory genes might also have been retained by the transformed cells and could play a role in regulating the tk gene in nonsuperinfected cells. The transformed cell line used (designated H2) was isolated by Davidson et al. (5) after the exposure of Ltk- cells to UV-irradiated HSV type 1 (HSV-1), strain VR3. In previous studies it was shown that phenotypically tk- revertants could be isolated from this cell line (5) and that expression of tk in the phenotypically tk+ cells could be enhanced by superinfection with tk- HSV-1 (23). For this study, clonal derivatives of the H2 cell line were isolated and found to differ considerably in the levels of tk expressed. It was shown that tk activity in all the clonal derivatives could be enhanced by superinfection with tk- HSV and could be induced in a phenotypically tkclone. These and other results indicate that the absence of tk activity in the phenotypically tkclones is not due to changes in the endogenous viral tk gene with respect to sequences encoding the enzyme or sequences that may be recognized by viral products for induction of the gene. Experiments designed to determine whether viral regulatory factors were expressed in phenotypically tk+ nonsuperinfected cells gave negative results despite the fact that the H2 cell line has been shown to contain a large portion of the viral genome (Leiden, Ph.D. thesis), including regions believed to code for early a polypeptides (28, 33). Comparison of the phenotypically tkand tk+ cell lines revealed one difference, namely that the phenotypically tk+ cells were significantly more permissive for HSV replication than were the phenotypically tk- cells or untrans- J. VIROL. formed Ltk- cells. Factors responsible for the generally enhanced rate of viral protein synthesis and accumulation of infectious progeny after superinfection of phenotypically tk+ cells may be related to the factor responsible for expression of the endogenous tk gene in nonsuperinfected cells. MATERLALS AND METHODS Cells. Mouse L929 (L cells) were obtained from J. M. Moulder (University of Chicago). R. Davidson (Harvard Medical School, Boston, Mass.) kindly provided us with the tk-deficient line of mouse L cells (Ltk- cells) isolated by Kit et al. (18) and a transformed cell line (designated H2) that carries the HSV- 1 tk gene and was isolated after exposure of Ltk- cells to UV-inactivated HSV-1(VR3) (5). The L cells and Ltk- cells were passaged in Ham F1 medium supplemented with 1% inactivated fetal calf serum, penicillin (15 U/ml), and streptomycin (15,g/ml) (designated F medium). The H2 cell line was passaged in F medium supplemented with lo5 M hypoxanthine, 1.6 x 1' M thymidine, and 4.4 x 1-7 M methotrexate (HAT medium), a modification of Littlefield medium that was designed for the selection of cells expressing the tk+ phenotype (26). The previously described F medium, when supplemented with 3,ug of bromodeoxyuridine (BUdR) (B medium), was used for the selection of cells expressing the tk- phenotype. HEp- 2 cells, grown in Dulbecco's modification of Eagle medium, were used for the production of virus stocks and of infected cell lysates for immunization of rabbits. Viruses. A tk- mutant of HSV-1 (B26) and the tk+ parental strain (Cl 11) were obtained from S. Kit (Baylor University, Houston, Tex.). The properties of these viruses have been described previously (7, 15, 16). The HSV-1(VR3) strain was also used in some of the experiments. Stocks of each of these virus strains were prepared as previously described (37) from HEp- 2 cells that had been infected at a low multiplicity (.1 PFU per cell); the stocks were titered on Vero cell monolayers. Chemicals. Cycloheximide was obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio), actinomycin D was from Calbiochem-Behring Corp. (La Jolla, Calif.), BUdR was from Sigma Chemical Co. (St. Louis, Mo.), and polyethylene glycol-6 was from J. T. Baker Chemical Co. (Phillipsburg, N.J.). [2-14C]- thymidine (5 mci/mmol), [3H]thymidine (5 Ci/ mmol), and L-[3S]methionine (1 to 4 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, Mass.). All chemicals for electrophoresis, including acrylamide and the bisacrylamide and N,N'- diallyltartardiamide cross-linkers, were obtained from Bio-Rad Laboratories (Richmond, Calif.). Cloning procedure. Cell monolayers were dispersed by use of an EDTA-trypsin (.5%) solution in phosphate-buffered saline (PBS), and the cells were collected by centrifugation and then resuspended in F medium. After determining cell concentrations by use of a hemacytometer, the suspensions were diluted and distributed to 25-cm2 Corning culture flasks containing the appropriate medium. The culture flasks were incubated undisturbed for 2 weeks, at which time some

3 VOL. 37, 1981 were stained with Giemsa for the enumeration of colonies, whereas individual clones were picked from other flasks having three well-separated colonies or less. Only one clone was selected from each flask, after breaking open the top. Clones were isolated with stainless steel cloning rings, detached with an EDTA-trypsin solution, and then transferred in the medium used for cloning. Infection and radiolabeling of cells. Confluent monolayer cultures were exposed to the indicated input multiplicity of virus in a small volume of F medium and maintained with continuous shaking for 2 h at 37 C. After replacing the virus inoculum with fresh F medium, incubation of the cultures was continued at 37 C. Mock-infected cultures were treated exactly as described for infected cultures except that virus was omitted. The experimental conditions in which cycloheximide and actinomycin D were utilized during infection have been described previously (23). For the pulse-labeling of infected cells, monolayers were rinsed with methionine-deficient medium 199 supplemented with 1% fetal calf serum and then incubated at 37 C for 1 min in this same medium containing [3S]methionine at 1,uCi/ml. Immediately after the labeling period, the cells were washed with cold PBS and scraped into a solubilization solution consisting of 5 mm Tris-hydrochloride (ph 7.), 5% B8-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and a trace of bromophenol blue tracking dye. Samples were sonicated for 1 min and then heated in a boiling water bath for 3 min before electrophoresis. Assay and neutralization of tk activity. At various times after the infection or mock-infection of cell cultures in 25-cm2 flasks, the cells were scraped from the flask surface, collected by centrifugation, washed with cold PBS, and frozen at -7 C in a small volume of PBS. Within 24 h the cells were thawed and disrupted by freeze-thawing three times; the lysates were used without further treatment. Aliquots (1 pl) of these lysates containing approximately 4 mg of protein per ml were mixed with 1l,l of a solution containing.125 M phosphate buffer (ph 6.), 12.5 mm ATP, 2 mm NaF, 2 mm phosphocreatine,.5 U of creatine phosphokinase (14 U/mg of protein), and 2.5 MCi of ['4C]thymidine (nominally 5 mci/mmol) and incubated at 38 C for 15 min. Subsequent steps in the assay were performed as described previously by Leiden et al. (23). Enzyme activity is expressed as counts per minute of [14C]TMP formed (the average value of duplicate determinations) per microgram of protein in the crude cell lysate or per 16 cells. An antiserum that had neutralizing activity against viral tk was produced in a rabbit by multiple injections of lysates prepared from HSV-1(Cl 11)-infected HEp- 2 cells. For preparations of the lysates, the infected cells were suspended in PBS (2 x 18 cells per ml), sonicated, and then centrifuged for 1 h at 25, rpm in the SW 27.1 rotor. The supernatant fraction was mixed with complete Freund adjuvant for the first injection and with incomplete adjuvant for all subsequent injections. Neutralization of tk activity was performed by mixing 1 ll of crude cell lysate with 5 pl of serum. After 1 h at C, the mixture was assayed for residual tk activity as described above. Autoradiography of cell monolayers. Confluent HSV THYMIDINE KINASE IN TRANSFORMED L CELLS 461 cell monolayers, infected or mock-infected, were labeled by incubation from 2 to 8 h after infection with medium 199 containing [3H]thymidine at 1,uCi/ml. The cells were rinsed with cold PBS and fixed to the plate by exposure to a methanol-acetic acid solution (3:1) for 15 min followed by methanol for another 15 min. The dried plates were coated with a 65% (vol/ vol) solution in water of Kodak photographic emulsion (NTB-2), dried in a darkroom for 2 h, and then stored in a light-impermeable box for 3 days. The emulsion was developed by soaking in Kodak D-19 developer for 1 min, followed by a 1-min treatment with Kodak Rapid-Fix. Plates were rinsed with water, stained with Giemsa for 1 min, then rinsed and dried. PEG-mediated cell fusion. Culture flasks (25 cm2) were seeded with equal numbers of cells from two different cell lines as described in Results. After 24 h the confluent monolayers were rinsed with F medium containing no serum. This was followed by treatment with a 5% (vol/vol) solution of polyethylene glycol (PEG)-6 in F medium without serum for 45 s. The PEG was removed by a series of rapid rinses, each containing a decreasing percentage of PEG, and finally by two rinses with F medium alone. Cells were then incubated for 2 h in F medium containing 5% serum, during which time massive cell fusion occurred. At this time the medium was replaced with fresh F medium containing 1% calf serum. Plaque assays. Confluent monolayer cultures (25- cm2 culture flasks) were inoculated with an HSV-1(Cl 11) virus suspension in 1 ml of PBS, incubated at 37 C for 2 h, and then overlaid with medium 199 supplemented with 5% inactivated fetal calf serum and 1.5% agarose. Incubation of the cultures was continued at 37 C. After 3 days of incubation and at daily intervals thereafter, triplicate cultures were overlaid with fresh medium 199, 5% fetal calf serum,.5% agarose, and.1% neutral red dye. These cultures were maintained for an additional 12 h in the dark, at which time they were examined for the enumeration of plaques. Acrylamide gel electrophoresis. Thymidine kinase activities were fractionated by electrophoresis on acrylamide gel slabs and localized in the gel essentially as described by Tischfield et al. (4). Cell extracts were prepared by suspending PBS-washed cells in.1 M Tris-hydrochloride (ph 7.5), 2.5 mm MgC92, 7.5 mm ATP,.5% bromophenol blue tracking dye,.5% Nonidet P-4, and 1% glycerol at a concentration of approximately 5 x 17 cells per ml. After 1 min on ice, cell nuclei were removed by low-speed centrifugation. Aliquots (1 IL) of cytoplasmic extract were subjected to electrophoresis on acrylamide gel slabs for 3 h at 4 C and 35 ma per gel. The separating gel consisted of 5.5% acrylamide,.165% N,N'-methylenebisacrylamide,.4 M Tris-hydrochloride (ph 8.5), 2 mm MgCl2,.35% ammonium persulfate, and.6% TEMED (N,N,N',N'-tetramethylethylenediamine), whereas the stacking gel contained 5% acrylamide,.125% N,N'-methylenebisacrylamide,.1 M Tris-hydrochloride (ph 7.), 2 mm MgCl2,.7% ammonium persulfate, and.1% TEMED. The electrode buffer (25 mm Tris-hydrochloride,.192 M glycine, 2 mm MgCl2, ph 8.5) was supplemented with 2.5 mm ATP in the upper chamber only. At the termination of electrophoresis, the gel slab was placed in 2 ml of

4 462 BUTTYAN AND SPEAR reaction solution consisting of.1 M Tris-hydrochloride (ph 7.), 2 mm MgCl2, 12.5 mm ATP, and 5 pl of [2-'4C]thymidine for 9 min at 38 C. At this time the ["4C]TMP reaction product was fixed in the gels by soaking them overnight at 4C in.1 M Trishydrochloride, ph 7., containing.1 M LaCl3. Unphosphorylated thymidine was removed by washing the gels for 24 h in tap water. Finally, the gels were prepared for fluorography exactly as described by Bonner and Laskey (1). Acrylamide gel electrophoresis of SDS-denatured samples was done on 9% acrylamide slabs, cross-linked with diallyltartardiamide exactly as described by Heine et al. (9). RESULTS Isolation and characterization of clones from the LVtk+-transformed cells H2. The transformed cell line obtained from R. Davidson (H2) had been passaged and maintained in HAT medium for 3 years in our laboratory before the initiation of this project. Before cloning (Fig. 1), these cells were passaged once in nonselective F medium. Trypsinized cell suspensions were diluted and plated in F medium to yield approximately five clones per 25-cm2 culture flask. Ten individual clones were picked from different plates and replated to establish ten different cell lines (Fl through F1). Subsequent cloning of the cell line F1 in B medium enabled the isolation of a BUdR-resistant clone, F1O/B, which was amplified by growth in B medium to yield a new cell line. By sequential cloning of this line in selective media, alternating between HAT H2 Maintained continuously in HAT medium Passaged once in non-selective medium Cloned in non-selective medium F4 lfio Produces high levels Produces low levels of viral tk of viral tk Cloned in BUdR medium Cloned l - FIO/B-H in HAT- FIO/B Produces viral tk medium Negative for I viral tk Cloned in BUdR medium Cloned jffiob- J-in HAT- FIO/B-H-B-HI medlium Initially negative Produces viral tk for viral tkunstable phenotype FIG. 1. Protocol for the isolation ofclonal variants from the LVtk' parental line, H2. J. VIROL. medium and B medium, three additional cell lines were obtained (F1O/B-H, F1O/B-H-B and F1O/B-H-B-H) as indicated by the protocol outlined in Fig. 1. At each step in this procedure the HAT-resistant or BUdR-resistant clones were amplified to approximately 1.5 x 18 cells in HAT medium or B medium, respectively, before recloning in the other selective medium. All the derived cell lines were maintained in the medium used for isolation, and after passage for a period of approximately 25 cell generations, cell extracts were prepared from each for quantitation of tk activity. Also, at this time, each cell line was tested for its cloning efficiency in selective HAT and B media and in nonselective medium. Even after only one passage in nonselective medium, the H2 cell line yielded clones that differed markedly in the levels of tk activity and cloning efficiencies in selective media (Tables 1 and 2). A clone derived and maintained in B medium, F1O/B, demonstrated the lowest levels of tk activity, comparable to that found in extracts of Ltk- cells. The cell line obtained in HAT selective medium, F1O/B-H, had relatively high tk levels. The ability of these cell lines to form colonies in selective media correlated roughly with the relative levels of tk activity detected in cell extracts. Those isolates which expressed high levels of tk activity had proportionally high cloning efficiency in HAT medium and low cloning efficiency in B medium, whereas the opposite relationship was observed in those TABLE 1. Levels of tk activity and plating efficiencies in selective media of clones isolated under nonselective conditions from the H2 cell line tk activ- Relative cloning ity (cpm efficienciesa Cellline per 16 HAT/ BUdR/ cells x 1-3) nonselec- nonselective tive H2b Clones of H2 Fl F F F F F F F F F a For all cell lines, 5% to 9% of cells plated in the nonselective F medium were able to form colonies. bthis cell line, obtained from R. Davidson, was tested after passage for 3 years in HAT medium, in parallel with the cloned derivatives of H2.

5 VOL. 37, 1981 lines with low levels of tk activity in cell extracts (Tables 1 and 2). One apparent exception was cell line F2, which expressed a relatively high level of tk activity and yet formed colonies in B medium almost as efficiently as in HAT medium. In the studies described in this report, attention has been focused on only a few of these clones: F4, which had the highest tk levels in cell extracts, and the several derivatives of F1, in particular F1/B, which had the lowest detectable tk activity corresponding with a high fractional cloning efficiency in B medium. Induction of tk synthesis in phenotypically tk- transformed cells. Previous experi- TABLE 2. Levels of tk activity and plating efficiencies in selective media of clones isolated from F1 under selective conditions Relative cloning tk activity efficiencies' Cell line (cpm/pg of protein HAT/ BUdR/ x 1-2) nonselec- nonsetive lective Ltk-.18 < Clones of H2 F F Clones of F1 F1/B F1/B-H F1/B-H-B NTb.8.64 a See footnote to Table 1. b NT, Not tested To F4 HSV THYMIDINE KINASE IN TRANSFORMED L CELLS 463 ments (23) have shown that superinfection of H2 cells with tk- HSV-1 can enhance the levels of viral tk detected in cell extracts. As expected, when the LVtk+ cell lines F4 and F1O/B-H were superinfected with tk- HSV-1 at 2 PFU per cell, there was a significant enhancement of the tk activity in cell extracts (Fig. 2). Similar results were obtained with all the cloned derivatives listed in Table 1 (data not shown). The new finding to emerge from the results shown in Fig. 2 is that superinfection of the phenotypically tkclone, F1O/B, resulted in induction of tk synthesis Ṫwo types of experiments indicated that the tk enzyme induced by superinfection of the F1/ B cell line was viral in origin. First, a hyperimmune rabbit antiserum produced against tk+ HSV-1-infected HEp-2 cells effectively neutralized tk activity present in extracts of the clonal isolates, F4 and F1O/B-H, and also the tk activity induced in the F1O/B line by superinfection (Table 3). This antiserum had little, if any, effect against tk activity present in extracts of uninfected F1O/B cells, Ltk- or normal tk+ L929 cells, yet neutralized almost all tk activity in extracts of Ltk- cells infected with tk+ HSV-1. Thus, the enzyme induced in the F1O/B line by superinfection with tk- HSV-1 had the antigenic properties of HSV-1 tk. Second, cytoplasmic extracts of the various cell lines, infected or uninfected, were subjected to electrophoresis in nondenaturing acrylamide gels and then the gels were processed as described in Materials and Methods to locate the bands of active tk's. The electrophoretic profiles g 2 - Hours after infection FIG. 2. Induction of tk activity in phenotypically tk+ (F4 and F1/B-H) or tk- (FlOIB) cell lines derived from the transformed cells H2. Extracts were prepared for assay at the indicated times after infection with tk- HSV-1 (-) or following mock-infection (). The multiplicities ofinfection found to be optimal for enhancement of tk activity and used in these experiments were 2 PFUper cell for the tk+ cells and 5 PFUper cell for the tkcells. In this and all subsequent figures tk activity is expressed as counts per minute of phosphorylated [14C]thymidine generated per microgram ofprotein after 15 min of incubation.

6 464 BUTTYAN AND SPEAR TABLE 3. Antigenic specificity of tk expressed by infected or uninfected cells tk activity (cpm Ratio of per 5,pl of reac- activity tion mixture) (anti- Cells Virus HSV-1/ Preim- Anti- HSVmune HSV-1 preimserum serum mune) F1O/B 5 PFU of 16,34 2, tk- HSV- 1 per cell F1O/B None 2,64 2, F4 None 9,653 1,896.2 F1O/B-H None 24,965 2,43.1 Ltk- 1 PFU of 18,489 3,52.3 tk+ HSV- 1 per cell Ltk- None 1,488 1, L929 None 2,659 2, of the tk activities detected are shown in Fig. 3. All cell lines possessed a fast-migrating band of activity, presumably mitochondrial tk since this is the only band present in extracts of Ltk- and F1O/B cells. Normal L929 cells produced an extremely slow-migrating band of activity representative of the normal mouse cell cytoplasmic enzyme. Ltk- cells infected with tk+ HSV-1 and the tk+ transformed clones, F4 and F1O/B-H, expressed a tk activity of intermediate mobility, different from those present in normal or uninfected mouse L cells. The tk that appeared in extracts of FlO/B cells after superinfection with tk- HSV-1 had a mobility indistinguishable from that of the virus-related band in tk+ transformants or in the cells infected with tk+ HSV-1. To determine what fraction of F1O/B cells was induced to produce viral tk after superinfection, F4, F1O/B, and Ltk- cultures were incubated for 6 h with medium containing [3H]thymidine (1,uCi/ml), beginning 2 h after infection with tk- HSV-1 or 2 h after mock infection. The labeled monolayers were then fixed to the culture dishes, overlaid with a photographic emulsion, and processed as described in Materials and Methods. Figure 4 shows that, in mock-infected F1/ B cultures, few if any of the cells were autoradiographically positive for the incorporation of [3H]thymidine. In the F1O/B cultures infected with tk- HSV-1, however, approximately 3 to 4% of the cells had incorporated thymidine into nuclear macromolecules, resulting in the presence of developed grains. This indicates that a large fraction of the F1O/B cells are subject to the induction of viral tk activity after superinfection. We have not been able to significantly increase the fraction of autoradiographically positive F1O/B cells by varying the time or duration of exposure to [3H]thymidine. Moreover, increasing the multiplicity of infection would probably not increase the fraction of positive cells since 5 PFU per cell was found to be optimal for inducing tk activity detectable in extracts of F1O/B cells. Reasons for the apparent inability to "turn on" all the F1O/B cells for tk expression could include resistance of a fraction of the cells to superinfection and heterogeneity of the cells with respect to retention of a reactivable tk gene or response to the viral products that mediate tk induction. It is interesting to note from Fig. 4 that a significant fraction of the uninfected F4 cells (tk+) remained unlabeled under the condi-,..a, A -. a 4 J. VIROL. FIG. 3. Fluorograms ofacrylamide gel slabs showing bands of tk's detected as described in the text. The extracts analyzed were prepared from: (lane 1) Ltk- cells infected at 1 PFU per cell with tk+ HSV- 1; (lane 2) LVtk+ (F4) cells; (lane 3) Ltk- cells; (lane 4) L929 cells; (lane 5) Ltk- cells; (lane 6) Ltk- cells infected at 1 PFUper cell with tk+ HSV-1; (lane 7) LVtk+ (F4) cells; (lane 8) LVtk+ (F4) cells infected at 5 PFUper cell with tk- HSV-1; (lane 9) LVtk- (FlO1 B) cells infected at 5 PFU per cell with tk- HSV-1; (lane 1) LVtk- (FIOIB) cells.

7 VOL. 37, 1981 HSV THYMIDINE KINASE IN TRANSFORMED L CELLS 465 W_ & 4v, t -.L't. -1 * 4w Ev*-! #' {. i *-,t A 2 (1 FIG. 4. Radioautography of cell monolayers that were uninfected or infected as indicated, and then incubated in medium containing [3H]thymidine from 2 to 8 h after infection. The cells are LVtk' (F4) in panels A and D, LVtk- (FlOIB) in panels B and E, and untransformed Ltk- in panels C and F. The cells in panels A, B, and C were infected with tk- HSV-1 at 5 PFUper cell, whereas the cells in panels D, E, and F were mock-infected. tions used and that superinfection reduced the size of this fraction but did not entirely eliminate it. We have never succeeded in labeling 1% of H2 cell populations or of the cloned sublines, even under conditions of incubation with [3H]thymidine that result in labeling 1% of normal L929 cells (unpublished data). The reasons for these findings are not yet understood. Attempts to detect regulatory factors in the tk+ transformed celis. Because the synthesis of viral tk in cells productively infected with tk+ HSV-1 requires the prior synthesis of a proteins (8, 11, 32) and because expression of an a protein appears to be necessary for the enhancement of resident viral tk activity after superinfection of LVtk+ cells (19, 23), the possibility existed that HSV-1 a proteins are synthesized in the nonsuperinfected transformed cells and could play some role in regulation of the resident viral tk gene. To test this possibility, we devised a means of detecting a regulatory product(s) that could induce tk synthesis from a superinfecting tk+ HSV-1 genome under conditions in which the superinfecting virus was blocked in its own ability to mediate the transition from a to 18 protein synthesis. Previous studies have shown that the sequential treatment of HSV-1-infected cells with cycloheximide from the time of virus addition, and then with actinomycin D on removal of the cycloheximide, prevents the synthesis of tk (8) and, in fact, permits the synthesis of a mrna's and proteins only (11, 14, 36). The results presented in Fig. 5 confirm these observations and

8 466 BUTTYAN AND SPEAR also show that tk can be made under these restrictive conditions provided that early viral products are already present in the cells at the time of infection with the tk+ HSV-1. For the reconstruction experiment shown in Fig. 5, replicate cultures of Ltk- cells were infected with tk- HSV-1 in the absence of inhibitors and incubated for 3 h to allow the accumulation of early viral products. These cultures, along with other uninfected cultures of Ltk- cells, were then infected with tk+ HSV-1 in the presence of cycloheximide at a concentration (5 yg/ml) sufficient to reduce protein synthesis to 1.5% of control levels. At 4 h after infection with the tk+ virus, the cycloheximide was removed and in some cultures was replaced with actinomycin D at a concentration (1,tg/ml) sufficient to reduce [3H]uridine incorporation to 1.5% of control levels. The results show that tk activity was induced in the cultures preinfected with tk- HSV-1, whether or not actinomycin D was N x >1 Q.._ c c ) C E F 1.o.8F.6F.4 F Hours after cycloheximide removal FIG. 5. tk activity ofreplicate cultures of Ltk/ cells after infection with tk+ HSV-1 at 1 PFUper cell in medium containing cycloheximide (5 ug/ml). Cycloheximide was withdrawn at 4 h after infection concomitant with the addition of medium containing actinomycin D (1 Jg/ml) (OH, ) or medium without inhibitor (, O). Some of the cultures (, ) were preinfected with tk- HSV-1 at 2 PFUper cell, in the absence of inhibitors, 3 h before the infection with the tk+ HSV-1. J. VIROL. added at the time of cycloheximide removal, whereas no induced tk activity could be detected in the cultures infected only with tk+ HSV-1 in the presence of cycloheximide followed by actinomycin D. Thus, in the doubly infected cultures, early products supplied by the tk- virus enabled functional tk mrna to be synthesized from the tk+ viral genome during the 4-h cycloheximide block that prevented the expression of a genes from the tk+ genome. The delayed appearance of tk in the singly infected cultures treated only with cycloheximide reflects the time required for early viral protein synthesis and then for new transcription in the presence of viral proteins, as is required for the production of functional tk mrna (8, 11, 12, 32, 36). By using the same approach for detection of regulatory factors in the transformed cells, F4 cells and untransformed Ltk- cells were infected with tk+ HSV-1 at 1 PFU per cell in the presence of cycloheximide, and then upon removal of the cycloheximide at 4 h after infection, actinomycin D was added to some of the cultures but not to others. At the various times indicated, cell samples were extracted and assayed for tk activity (Fig. 6). For both the LVtk+ and Ltkcells treated sequentially with cycloheximide and actinomycin D after infection, the levels of tk activity remained unchanged throughout the experiment. In those cultures treated with cycloheximide alone, there was an induction of tk synthesis, albeit delayed. These results are consistent with previous findings (8, 23) that sequential treatment with cycloheximide and actinomycin D after infection prevents the expression of HSV tk either from the viral genome or from the tk gene resident in transformed cells, whereas cycloheximide alone only delays the induction. The finding pertinent to this study is that LVtk+ cells contained no factors capable of inducing tk mrna synthesis from the superinfecting viral genome during the cycloheximide block. Figure 6 also shows that the level of tk activity remained unchanged in uninfected F4 cells that were treated sequentially with cycloheximide and actinomycin D. Other infected cultures of F4 cells and Ltkcells were pulse-labeled with [35S]methionine at various times after release of the cycloheximide block in the presence or absence of actinomycin D to monitor the synthesis of proteins in the infected cells. As shown by the electropherograms in Fig. 7, neither the infected Ltk- cells nor infected F4 cells produced detectable levels of polypeptides previously assigned to the 18 or y classes (1, 11) in the presence of actinomycin D, although all classes of polypeptides were made after removal of cycloheximide in the ab-

9 cm 7-6 Ltk- cel Is F4 cells C C Hours after infection FIG. 6. tk activity of LVtk+ (F4) or Ltk- cells at the indicated times after infection with tk+ HSV-1 at 1 PFU per cell in medium containing cycloheximide (5 pg/ml). Cycloheximide was withdrawn at 4 h after infection (indicated by the arrow) followed by the addition of medium containing actinomycin D (1 pg/ml) () or medium without inhibitor (). Also shown is the thymidine kinase activity of uninfected F4 cells subjected to sequential treatment with cycloheximide (5 ug/ml) for 4 h followed by actinomycin D (1 pg/ml) (O). F4 Ltk- Cyclo., Cyclo. then act. D cyl.cyclio., Cyclo.then act. D A I. QAA -' WA2U< z 5 FIG. 7. Autoradiogram of a sodium dodecyl sulfate-acrylamide gel slab after electrophoresis of [35S]- methionine-labeled polypeptides solubilized from LVtk+ (F4) or Ltk- cells at the designated times after infection with tk+ HSV-1. The cells were infected at 1 PFUper cell in medium containing cycloheximide at 5 pg/ml. The cycloheximide was withdrawn at 4 h after infection followed by the addition of medium containing actinomycin D (1 plg/ml) or medium without inhibitor as indicated. The cultures were pulselabeled with [35Slmethionine for 15 min immediately before harvest and solubilization of the polypeptides. Each well was loaded with 25 ul of sample obtained from 2.5 x 15 cells. Some of the most abundant viral polypeptides are labeled according to the designations of Honess and Roizman (1, 11) who showed that polypeptides 4 and are representative of the a class, polypeptides 6 and 8 are representative of the f, class, and polypeptide 5 is representative of the y class. 467

10 468 BUTTYAN AND SPEAR sence of actinomycm D. It was noted in this experiment that all the viral polypeptides were synthesized earlier and in larger quantities in the F4 cells than in Ltk- cells. This phenomenon is explored further in a later section. These results presented in Fig. 6 and 7 suggest that little if any HSV a regulatory proteins are endogeneously available in the LVtk+ cells to act on the genome of a superinfecting HSV-1, at least with respect to the immediate induction of tk or other fi or y polypeptides that can be detected by electrophoresis. The use of polyethylene glycol as a cell fusing agent allowed us to investigate the possibility that diffusible factors of either viral or cellular genetic origin may influence expression of the resident viral tk gene in the transformed LVtk+ or LVtk- cells. Cell monolayers prepared by seeding 1:1 mixtures of transformed cells (F4 alone or F4 and F1O/B) or transformed and untransformed cells (F4 and Ltk- or F1O/B and Ltk-) were treated with a 5% (vol/vol) solution of PEG-6 in F medium for a brief period, resulting in massive cell fusion within 2 h after exposure to the PEG. At regular intervals after PEG treatment, extracts made from the fused cells were tested for tk activity. The results, 9 F4: F4 J. VIROL. presented in Fig. 8, show that tk activity in the fused mixture of F4 and F1O/B cells was intermediate between the levels observed for the fused F4 cells and for the fused mixtures of F1/ B and Ltk- cells. This activity remained constant for a period of at least 14 h after treatment with PEG. Thus, we found no indication for the presence of diffusible regulatory factors, either positive or negative, in the transformed cells. Enhanced expression of superinfecting HSV-1 in phenotypically tk+ transformed cells. The LVtk+ and LVtk- transformed cell variants were tested for their ability to support wild-type HSV replication. F4 cells, F1O/B cells, or Ltk- cells were infected with tk+ HSV-1 at 2 PFU per cell. At various times after infection, cultures were harvested, and the number of infectious units was determined by titration on Vero cell monolayers. As seen in Fig. 9, progeny virus was detected earlier and significantly higher yields of virus were produced in the infected F4 cells than during an equivalent infection of F1O/B or Ltk- cells. An experiment was designed to determine whether this effect was due to greater yields of HSV progeny per F4 cell or to differential adsorption or penetration of the virus among the different cell lines. Con- 8 N 1-7 x, 6._ U 9 5 C._4 S C I F4; Ltk- F4: FIO/B Ltk-: FIO/B U, n ccf E._ 17 X16 a- I Hours after PEG treatment FIG. 8. tk activity in extracts prepared at the indicated times after exposure of mixed cell cultures to PEG. The cultures were seeded as 1:1 mixtures of the indicated cell lines. As early as 2 h after the PEG treatment, massive polykaryocyte formation was evident in all cultures. I O Hours after infection FIG. 9. Yield of progeny HSV-1 at the indicated times after infection of F4 cells (-), F1/B cells (A), or Ltk- cells () with tk+ HSV-1 at 2 PFU per cell.

11 VOL. 37, 1981 fluent monolayers of LVtk+, LVtk-, and Ltkcells were infected with aliquots of a diluted tk+ HSV-1 stock and then overlaid with medium containing agarose. At intervals after infection, medium containing neutral red and agarose was added to replicate cultures. Visible plaques were counted 12 h later. As seen from Fig. 1, HSV-1 ultimately formed the same number of plaques on monolayers of LVtk+ (F4 or F1/B-H), LVtk- (F1/B or F1/B-H-B) or Ltk- cells, indicating that infection was initiated equally efficiently on all three cell types. Visible plaques were detected much earlier and grew larger on monolayers of LVtk+ cells, however, suggesting greater yields of viral progeny per cell. Analysis of HSV-1 viral protein synthesis in the various cell lines was done by pulse-labeling infected cell cultures with [35S]methionine at different times after infection. The labeling patterns shown in Fig. 11 demonstrate that proteins representative of each of the HSV viral polypeptide classes (a, /3, and y) were synthesized at earlier times in the LVtk+ cells than in LVtk- or Ltk- cells. In addition, host protein synthesis was inhibited earlier in LVtk+ cells. DISCUSSION In a previous publication from this laboratory (23), it was shown that the level of tk activity expressed from the resident viral tk gene in H2 cells could be enhanced by superinfection with HSV THYMIDINE KINASE IN TRANSFORMED L CELLS 469 tk- HSV-1. Similar findings have been reported for other cell lines transformed by UV-inactivated HSV (17, 25). Other studies of H2 cells have indicated that resident viral tk gene expression can be turned off and then on again in successive generations and is therefore somehow modulated in the absence of superinfection (5). By isolating clones from the H2 transformed cell line that express different levels of viral tk and by showing that viral tk activity can be induced in phenotypically tk- clones after superinfection with tk- HSV-1, we have used a different approach to confirm the previous finding (5) that transformed cells may retain a potentially reactivable HSV tk gene without expressing it (LVtk- cells). It is likely that expression of the viral tk gene in the transformed cells is regulated by one or both of two general mechanisms: (i) physical arrangement of the DNA sequences coding for the viral tk polypeptide, its promotor(s), or other signals needed to generate functional mrna or (ii) availability of a specific regulatory factor(s) which can affect either the transcription, posttranscriptional processing, or translation of the viral tk gene. With regard to the first general mechanism, our finding that tk activity can be induced in a significant proportion of the phenotypically tk- cells, F1/B, rules out rearrangements in the DNA sequence encoding the tk polypeptide as an explanation for the tk- phe- 4 - A *, 3-2 / Days after plating FIG. 1. Numbers ofplaques detectable at the indicated times after plating identical samples of tk+ HSV- 1 on the tk+ transformed cells, F4 () and FJO/B-H (-), the phenotypically tk- transformed cells, F1B (A) and F1O/B-H-B (V), or on Ltk- cells (). Neutral red was added to the agarose overlay medium 12 h before the enumeration ofplaques.

12 47 BUTTYAN AND SPEAR J. VIROL. F4 F 1O/B Ltk j1o FIG. 11. Autoradiogram of SDS-acrylamide gel slabs after electrophoresis of [5S]methionine-labeled polypeptides solubilized from F4 cells, F1/B cells, or Ltk- cells at the indicated times after infection of the cells with tk+ HSV-1. The cells were infected at 2 PFUper cell and were pulse-labeled with /js]methionine for 1 min immediately before solubilization of the polypeptides for analysis. Each well was loaded with 25,ul of sample obtained from 2.5 x 15 cells. Only the most abundant viral polypeptides are labeled, according to the designations of Honess and Roizman (1, 11); see also the legend to Fig. 7. notype, unless the rearrangements can be specifically reversed by superinfection with tk- HSV-1. The possibility exists, however, that rearrangements in non-tk-coding DNA sequences required for transcription of the endogenous viral tk gene or processing of transcripts could account for the tk- phenotype. A necessary corollary of this hypothesis is that regulatory DNA sequences required for expression of the gene in nonsuperinfected cells are different from those required for expression of the gene after superinfection, because tk activity was induced after superinfection of the phenotypically tk+ or tk- cells. If we assume that the resident viral tk gene is integrated into host DNA sequences, it is possible that transcription of the viral tk gene can be promoted from two sequences, perhaps one of viral origin and the other on contiguous cellular DNA sequences. Assuming also that the products of superinfecting virus are acting on a viral promotor sequence, this sequence must be intact in both the tk+ and tk- cells. With regard to the second possible mechanism of resident viral gene regulation, if there are specific regulatory factors present in either the LVtk+ or LVtk- cells, they are not readily diffusible from nucleus to nucleus in heterokaryons. This was shown by the absence of any detectable change in resident tk activity after fusion of LVtk+ and LVtk- cells induced by polyethylene glycol. We also tested the possibility that the product of a viral regulatory gene, perhaps incorporated into the transformed cells coincidentally with the viral tk gene, could be a factor in regulation of the resident viral tk gene (23). Experiments designed to test this hypothesis showed that LVtk+ cells could not provide the viral regulatory products necessary to permit expression of tk or other f8 or y polypeptides from a superinfecting viral genome. Yet the expression of tk by tk+ cells (both F4 and F1/ B-H) correlated with greater permissivity of these cells for tk+ HSV replication than was observed with LVtk- cells or untransformed Ltk- cells. It seems unlikely that the levels of viral tk available before infection could explain these results because the superinfecting virus should supply all the enzyme required. The possibility exists that some other factor expressed in LVtk+ cells (either of viral or cellular genetic origin) can enable enhanced expression of superinfecting viral genomes as well as of the resident viral tk gene. Alternatively, a cellular factor expressed in LVtk- cells and Ltk- cells, but not in LVtk+ cells, may have a negative influence on the expression of HSV genes. ACKNOWLEDGMENTS This work was supported by Public Health Service grants

13 VOL. 37, 1981 from the National Cancer Institute (2 P1 CA and 5 R1 CA-21776). R. B. was a trainee supported by Public Health Service training grant 1 T32 AI-7182 from the National Institute of Allergy and Infectious Diseases. P.G.S. is recipient of Research, Career Development Award 5 K4 CA-35. LITERATURE CITED 1. Bonner, W. M., and R. A. Laskey A film detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46: Chadha, K., and W. Munyon Presence of herpes simplex virus related antigens in transformed L-cells. J. Virol. 15: Chadha, K. C., W. H. Munyon, and R. F. Zeigel Expression of type-specific virion structural antigen(s) in L-cells transformed by herpes simplex virus types 1 and 2. Virology 76: Clements, J. B., R. J. Watson, and N. M. Wilkie Temporal regulation of herpes simplex virus type 1 transcription: location of transcripts on the viral genome. Cell 12: Davidson, R., S. Adelstein, and M. 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