Control of Expression of the Vaccinia Virus Thymidine Kinase

JOURNAL OF VIROLOGY, Nov. 1981, p. 456-464 0022-538X/81/110456-09$02.00/0

Vol. 40, No. 2

Control of Expression of the Vaccinia Virus Thymidine Kinase Gene DENNIS E. HRUBY* AND L. ANDREW BALL Biophysics Laboratory, Graduate School, and Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 26 May 1981/Accepted 9 July 1981

mRNA extracted from vaccinia virus-infected cells early after infection directs cell-free synthesis of enzymatically active viral thymidine kinase (Hruby and Ball, Virology, in press). We used this assay for a specific vaccinia virus mRNA to study the induction and repression of the viral thymidine kinase gene during infection of thymidine kinase-deficient L-cells. As observed previously by other workers, the synthesis of thymidine kinase occurred immediately after infection but was switched off about 4 h later. We observed similar kinetics of accumulation and shutoff under conditions where viral DNA synthesis and late gene expression were inhibited. Cell-free translation of mRNA from infected cells showed that the concentration of functional message for viral thymidine kinase reached a peak 3 to 4 h after infection and then decreased with a half-life of about 1 h. These kinetics indicated that significant levels of thymidine kinase mRNA persisted in cells which had stopped synthesizing the enzyme. Under conditions where late gene expression was inhibited, high concentrations of functional mRNA could be isolated from cells at late times after infection. On the basis of these results, we conclude that the repression of thymidine kinase expression is mediated at the translational level by one or more early or delayed early viral genes. Repression is accompanied by, but does not depend on, the inactivation or degradation of thymidine kinase mRNA, which is a late gene function.

Vaccinia virus (VV), a member of the poxvirus group, is a large DNA-containing virus that replicates predominantly in the cytoplasm of infected cells (24). As well as facilitating the study of virus-specific events, this cytoplasmic site of replication has some important consequences. First, it means that VV virions must contain or encode many of the enzymes involved in viral DNA and RNA syntheses and maturation. Second, the mechanisms that control VV gene expression may differ substantially from those that regulate the expression of nuclear genes of both cellular origin and viral origin. For these reasons, we undertook a study of VV gene expression, concentrating initially on the viral thymidine kinase gene (tk gene). Like herpesvirus (25), VV contains a viral tk gene which is not essential for virus replication in cell culture and hence is amenable to genetic manipulation. Since expression of this gene is temporally regulated in vivo, the VV tk gene seemed to be an appropriate model for detailed studies. The VV genome is a linear double-stranded DNA molecule which has a molecular weight of about 122 x 106 (about 185 kilobase pairs), and more than 90% of the VV genome sequences are unique (8). Restriction enzyme analysis, molec-

ular cloning, and translational mapping techniques have revealed the approximate locations of the genes encoding many of the 150 to 200 virus-specific polypeptides (1, 2). However, the tk gene, which is thought to code for a polypeptide having an apparent molecular weight of 42,000 (16), has not been mapped yet. Expression of the genes of this virus is under strict temporal regulation during infection (7, 27), but the control mechanisms remain largely obscure. Although there are many different kinetic classes, VV genes are usually categorized as immediate early, delayed early, and late on the basis of sensitivity to inhibitors of viral RNA synthesis, protein synthesis, and DNA replication, respectively (24). As shown by McAuslan et al. (1923), the poxvirus tk gene is an immediate early gene since viral thymidine kinase (EC 2.7.1.75) was detected soon after infection and since the expression of this gene did not depend on the protein product of any other viral gene. Thymidine kinase activity increased linearly for 3 to 4 h after infection, but then further enzyme accumulation ceased. The timing of tk gene repression coincided with late viral gene expression. From studies on the expression of cowpox and rabbitpox tk genes in infected HeLa cells,

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McAuslan concluded that the repression mechanism required the synthesis of a dominant, trans-acting factor that was encoded in a late viral gene. Since drug-mediated inhibition of late gene expression resulted in the continued accumulation of thymidine kinase, he further concluded that the tk gene mRNA was functionally stable and that the repression mechanism operated at the translational level. However, different results were obtained with other poxviruscell systems; for example, in VV-infected L-cells tk gene repression seemed to be an immediate or delayed early function (3), whereas in HeLa cells infected with the CL-R strain of VV, no tk gene repression occurred (23). To elucidate the molecular mechanisms responsible for regulating VV tk gene expression during infection, we developed an assay for VV tk mRNA that is based on the translation of this mRNA into active enzyme in a reticulocyte lysate. In this paper we describe the use of this assay to measure the intracellular concentrations of functional tk mRNA both during uninhibited infection of mouse L-cells with VV and under inhibited conditions, where not all classes of viral genes could be expressed.

457

time of addition until the infection was terminated. Preparation of cell extracts for thymidine kinase assays. To monitor VV tk gene expression, the medium was removed from duplicate sets of confluent monolayers (60 by 15 mm) of Ltk- cells. The monolayers were washed twice with phosphate-buffered saline containing 1 mM magnesium chloride at 25°C, and then they were infected with purified VV (10 PFU/cell) in 0.5 ml of phosphate-buffered saline containing 1 mM magnesium chloride at 25°C for 30 min. After virus adsorption, the inocula were removed and replaced with 4 ml of medium at 37°C, and the cells were incubated at 37°C. At different times postinfection, cells in duplicate dishes were loosened by scraping with a rubber policeman, pooled, and harvested by centrifugation at 2,000 rpm for 5 min at 4°C in a Sorvall GLC-2B centrifuge. The cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM magnesium chloride and then lysed in 2 volumes of extraction buffer (0.5% Nonidet P-40 [BDH Chemicals Ltd.], 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.6, 2 mM magnesium acetate, 1 mM dithiothreitol, 50 MM thymidine). The lysates were clarified by centrifugation at 2,000 rpm for 10 min, and the resulting supernatants were frozen for subsequent thymidine kinase assays. Isolation and purification of W mRNA. Suspension cultures (about 500 ml) of Ltk- cells at a concentration of 5 x 105 cells per ml were harvested by centrifugation at 2,000 rpm for 10 min at 25°C in a Beckman J-6 centrifuge and then suspended at a conMATERIALS AND MEIHODS centration of 1 x 107 cells per ml in adsorption medium Virus and cells. VV strain WR was grown in HeLa (10). Purified VV (10 PFU/cell) was added, and the cells maintained at a concentration of 5 x 105 cells per cells were swirled gently at 37°C for 30 min. The ml in suspension cultures in Eagle minimal essential infected cells were then diluted to a concentration of medium supplemented with 10% calf serum. At 48 h 5 x 105 cells per ml and shaken at 100 rpm at 37°C for after infection (multiplicity of infection, 1 PFU/cell), the duration of the infection. For the experiment intracellular virus was released by lysing the infected shown in Fig. 6, confluent monolayer cultures of Ltkcells, and this virus was purified by sucrose gradient cells on 100-mm plates were infected with VV (10 centrifugation, as described previously (10, 11). The PFU/cell) as described above. At different times after titer of the purified virus was determined by plaque infection, the cells from five plates were harvested as formation on confluent monolayers of BSC-40 cells described above and pooled. Pellets of uninfected or (10, 11). Mouse L-cells that lacked thymidine kinase VV-infected Ltk- cells were washed three times with (Ltk- cells [15]) were kindly provided by R. Hughes, ice-cold phosphate-buffered saline containing 1 mM Roswell Park Memorial Institute. These cells were magnesium chloride. The cells were swollen in hypomaintained as either monolayer cultures or suspension tonic buffer (10 mM HEPES, pH 7.6, 10 mM NaCl, 1 cultures in the appropriate minimal essential medium mM magnesium chloride) and then broken with 20 containing 10%1o heat-inactivated fetal calf serum and strokes in a tight-fitting Dounce homogenizer. The the following supplements: 100MlM nonessential amino homogenate was centrifuged at 2,000 rpm and 4°C for acids, 16 ,uM thymidine, 50 uM adenosine, 50 yM 10 min. The resulting supernatant was removed, and guanosine, 10 MuM glycine, and 20 ,ig of 5-bromo-2'- the pellet was homogenized and centrifuged. The sudeoxyuridine per ml. All media and sera were obtained pernatants were pooled, made 2% (wt/vol) in N-laufrom GIBCO Laboratories. rylsarcosine and 1 g/ml in cesium chloride, and We used the following drug treatment protocols to warmed to 25°C. This mixture was layered over a 2block VV replication at desired steps: 5 Mg of actino- ml cushion of 5.7 M cesium chloride-0.1 M EDTA (in mycin D (Sigma Chemical Co.) per ml, added 2 h after hypotonic buffer containing 2% N-laurylsarcosine). infection; 100 Mg of 5,6-dichlororibofuranosylbenzim- Then the mixture was overlaid with hypotonic buffer idazole (Calbiochem) per ml, added 1.5 h after infec- (9). Centrifugation was in a Beckman SW40 rotor at tion; 25 MLg of cytosine arabinoside (Calbiochem) per 25,000 rpm and 25°C for 12 to 18 h with a Beckman ml, added 1.5 h before infection; 5 mM hydroxyurea model L5-50B ultracentrifuge. The resulting RNA pel(Sigma), added 1 h before infection; 10MlM 5-fluoro-2'- let was dissolved in sterile water and ethanol precipideoxyuridine (FUdR; Sigma), added at the time of tated twice in the presence of 0.15 M sodium acetate infection; and 100,ug of cycloheximide (Sigma) per ml, (pH 5.3). The mRNA was selected by affinity chroadded at the time of infection. The drugs were main- matography on oligodeoxythymidylic acid cellulose tained at the concentrations indicated above from the (grade T-3; Collaborative Research, Inc.) (12). Poly-

458

HRUBY AND BALL

adenylic acid-containing RNAs were eluted, ethanol precipitated twice, and stored at -20°C until they were used for translation. Reticulocyte lysate reactions. Fresh rabbit reticulocytes were purchased from Green Hectares, Oregon, Wis., and were used to prepare reticulocyte lysates (29). In a final volume of 15 Al, the cell-free translation reaction mixtures contained the following: 50% nuclease-treated reticulocyte lysate, 0 to 130 mg of VV mRNA per ml, 10 mM Tris-hydrochloride (pH 8.2), 25 jg of creatine phosphokinase (Sigma) per ml, 1.6 ,LM hemin, 125 mM potassium acetate, 0.6 mM magnesium acetate, 10 mM creatine phosphate (Calbiochem), 100 ,ug of calf liver tRNA (Boehringer Mannheim Corp.) per ml, a mixture of 19 amino acids (minus methionine) or 20 amino acids at a concentration of 100,uM each, 5 mM dithiothreitol, and 0.1 mCi of [3S]methionine (546.7 Ci/mmol; New England Nuclear Corp.) per ml. In addition, the reaction mixtures contained 4.5 mM cyclic AMP and 50 ,IM thymidine to prevent double-stranded RNA-mediated inhibition of protein synthesis (4) and to stabilize nascent thymidine kinase (5), respectively. Reaction mixtures were incubated at 300C for 2 h to monitor protein synthesis or for 4 h to synthesize active thymidine kinase. Protein synthesis was measured by spotting 2.5-pl samples of the reaction mixtures on Whatman 3MM filter disks and processing them to determine hot trichloroacetic acid-precipitable radioactivity, as measured by liquid scintillation counting. Polyacrylamide gel electrophoresis. Gel electrophoresis in the presence of sodium dodecyl sulfate was performed on 12.5% polyacrylamide slab gels by using the method of Studier (30). A 2-MlI portion of 35Slabeled cell-free translation products or an appropriate amount of 35S-labeled VV-infected cell lysate was diluted with 40 ,u of sample buffer, boiled for 5 min, and then electrophoresed at 150 V for 5 h. Autoradiography of stained dried gels was on Kodak X-Omat R medical X-ray film. Thymidine kinase assay. Extracts of VV-infected cells or reticulocyte lysates after 4 h of translation were assayed for thymidine kinase activity as follows. A 5-!d portion of extract was mixed with 3 pl of tk mixture (250 mM sodium phoshate, pH 6.0, 25 mM ATP, 25 mM magnesium acetate, 120 MM unlabeled thymidine, 1.3 mCi of [methyl-3H]thymidine [72 Ci/ mmol; New England Nuclear Corp.] per ml), and this mixture was incubated at 300C for 4 h. Under these conditions, the assay was linear with respect to time and amount of enzyme. After incubation, 40 1p of icecold water was added, and the reaction was terminated by heating at 1000C for 3 min. Denatured proteins were removed by 2 min of centrifugation in an Eppendorf model 5412 microfuge. Samples (40 pd) of supernatant were spotted onto 2.4-cm Whatman DE-81 filter paper disks, which were dropped immediately into 4 mM ammonium formate (pH 6.0) containing 10 ,uM thymidine at 370C. By using gentle agitation at 37°C, the filters were washed three times with ammonium formate-thymidine, once with water, and twice with 95% ethanol to remove non-phosphorylated thymidine (28). The filters were then dried and counted with a liquid scintillation counter at a counting efficiency of 5.3%. The values obtained by spotting

J. VIROL. a reaction mixture containing 5,ul of water and 3 ,ul of tk mixture were subtracted as background.

RESULTS Expression of VY thymidine kinase. To determine the kinetics of synthesis of VV thymidine kinase, we infected monolayers of mouse L-cells that were deficient in the cellular enzyme (Ltk- cells) with wild-type VV strain WR and monitored the thymidine kinase activity present in cytoplasmic extracts with time after infection (Fig. 1). Extracts of uninfected Ltk- cells showed essentially no activity, but during the first 4 h of VV infection thymidine kinase activity accumulated in the cytoplasm. After 4 h, no further activity accumulated, but the level remained constant for the next 18 h. The addition of cycloheximide to infected cells after 4 h postinfection produced no evidence of enzyme turnover (data not shown), so the plateau was attributed to a repression of active enzyme synthesis. Many lines of evidence from several groups of workers have indicated that this new enzyme is specified by the viral genome (24). The most persuasive evidence is that the new activity has electrophoretic and antigenic properties that are clearly distinct from those of the cellular enzyme (16) and that viral mutants which fail to induce the enzyme can be isolated (6). Working with rabbitpox and cowpox infections of HeLa cells, McAuslan found several ways to superinduce thymidine kinase by eliminating the repression mechanism (19-21). These 7 6

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Hours Post Infection

FIG. 1. Accumulation of thymidine kinase activity during VV infection. Monolayers of Ltk- cells were infected with wild-type VV(1OPFU/cell). At the times indicated, cell extracts were prepared and assayed for thymidine kinase activity. Symbols: 0, Ltk- cells; 0, VV-infected Ltk- cells; A, Ltk- cells infected with VV in the presence of actinomycin D (5 ,ug/ml) added 1.5 h after infection.

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VV

THYMIDINE KINASE EXPRESSION

459

2 3 4 5 6 7 8 9 10 11 12 included mild UV irradiation of the infecting virus, or prevention of late viral -ene expression a with inhibitors of viral DNA synthesis or inhibitors of RNA synthesis added after early tran-~I~ * -pe scription. For example, in the presence of FUdR rabbitpox thymidine kinase continued to accumulate for more than 16 h in HeLa cells rather -_ EEm-nm I_ than being switched off 4 h after infection. Similar results were observed in HeLa cells infected with VV strain WR (13). However, in L-cells infected with VV strain IHD, inhibition of late gene expression did not eliminate the repression Z-1 mechanism (31). Similarly, we found that the addition of actinomycin D 1.5 h after infection failed to delay significantly the shutoff of thymidine kinase accumulation in VV-infected Lcells (Fig. 1). An increase of 10 to 15% in the overall enzyme level was observed, but the kinetics of induction and repression closely resembled those of an uninhibited infection. Again, 2 3 4 5 6 7 8 9 10 11 12 the enzyme was stable. Similar results were ob- b tained when 5,6-dichlororibofuranosylbenzimidazole was added 1.5 h after infection as an alternative inhibitor of RNA synthesis and when hydroxyurea, cytosine arabinoside, or FUdR was added as an inhibitor of viral DNA synthesis. Viral polypeptide synthesis. Repression of early gene expression in poxvirus-infected cells has also been studied by [3S]methionine pulse-em-r ---.MWN- labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides of infected cells (7, 27). In VV-infected BSC-1 cells, the repression of early polypeptides did not occur when viral DNA synthesis was inhibited. This result is consistent with the observations of McAuslan of superinduction under these conditions, but it is less compatible with our finding of normal repression of thymidine kinase. Therefore, we analyzed the polypeptides that were FIG. 2. Sodiumdodecylsulfate-polyacrylamidegel synthesized in VV-infected L-cells both during uninhibited infection and under conditions electrophoresis of the polypeptides labeled in uninwhere viral DNA synthesis and late gene expres- fected and VV-infected L-cells. L-cells in suspension were infected with VV (10 PFU/cell) and sion were inhibited. Infected cultures were pulse- cultures were by incorporation of [3S]methiopulse-labeled labeled for 30-min periods throughout the first nine (10 for 30-min periods throughout the jtCi/ml) 6 h of infection either in the absence or in the first 6 h of infection. At the end of each labeling presence of FUdR, and the labeled polypeptides period, cells were harvested by centrifugation, and were resolved by electrophoresis on polyacrylthe resulting cellpellet was dissolved in sample buffer amide slab gels (Fig. 2). Upon infection, there containing sodium dodecyl sulfate. Polypeptides were subjected to electrophoresis on 12.5%polyacrylamide was an immediate change in the pattern of polypeptides synthesized, from a pattern of unin- slab gels and were visualized by autoradiography. fected L-cells to a pattern that was characteristic (a) Infection carried out in the absence of inhibitors. (b) Infection carried out in the presence of 10 pM of VV-infected cells at early times after infection FUdR. 1 and 12, Uninfected cells; lanes 2 (Fig. 2a). This change involved both the inhibi- through Lanes 11, VV-infected cells labeled for 30-min petion of cellular polypeptide synthesis and the riods starting at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, appearance of distinct new polypeptides, most and 6.0 h after infection. of which were presumed to be virus specified. The VV thymidine kinase is thought to be a to allow identification of what may be a very dimer of 40,000- to 42,000-dalton subunits (16), minor polypeptide species. In the uninhibited but the patterns shown in Fig. 2 are too complex infection, the pattern of polypeptide synthesis

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460 HRUBY AND BALL changed continuously during the first 6 h. Most of the early polypeptides were repressed, and several new species emerged, particularly after viral DNA synthesis, which occurred at 3 to 4 h after infection. The presence of FUdR did not greatly alter the pattern of polypeptides made at early times after infection (Fig. 2b). As Pennington observed (27), the inhibition of host protein synthesis was less severe in the presence of FUdR than in the absence of FUdR, and there were small differences in the relative amounts of some of the early viral polypeptides; however, in general, similar early genes were expressed in the presence and in the absence of FUdR. Moreover, as the infection proceeded, expression of several of these early genes, but not all of them, was repressed, despite the presence of FUdR and the consequent inhibition of viral DNA synthesis. The major effect of FUdR was the complete absence of all polypeptides except the early polypeptides that were detected immediately after infection. These results confirmed the conclusion that we reached based on the data shown in Fig. 1, namely, that in VV-infected L-cells the mechanisms responsible for repression of some of the early viral genes do not depend on late gene expression. Cell-free protein synthesis. To study the mechanism of repression in greater detail, we isolated mRNA's from cultures of uninfected and VV-infected Ltk- cells and used them to direct cell-free protein synthesis in mRNA-dependent reticulocyte lysates. Protein synthesis was monitored by the incorporation of [35S]methionine, and the polypeptide products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 3). mRNA from cells infected with VV for 2 h directed the synthesis of a large number of polypeptides, which had molecular weights ranging from less than 10,000 to more than 100,000 (Fig. 3, lanes 2 and 7). Many of the products had no counterparts among the polypeptides whose syntheses were directed by mRNA's from uninfected cells (lane 6), and thus these products were probably virus specific. In accordance with this and as shown by Cooper and Moss (4), many of these cell-free products comigrated with polypeptides synthesized in infected cells (data not shown). The syntheses of very similar products were directed by mRNA isolated from cells at 5 h after an infection carried out in the presence of cycloheximide (Fig. 3, lane 3). This drug stimulated and prolonged early VV transcription and prevented expression of the delayed early and late viral genes, so that this mRNA contained only immediate early transcripts. mRNA isolated from

J. VIROL. \

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FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in reticulocyte lysatesg in response to different mRNA preparations. Equal volumes of the reticulocyte lysate programmed with different mRNA's were subjected to electrophoresis on a 12.5%polyacrylamide slab gel, and the labeled polypeptides were visualized by autoradiography of the dried gel. Lane 1, No added mRNA; lanes 2 through 5, mRNA isolated fr-om Ltkcells infected with wild- type V V; lane 2, 2 h after an uninhibited infection; lane 3, 5 h after an infection carried out in the presence of cycloheximide; lane 4, 5 h after an infection carried out in the presence of hydroxyurea; lane 5, 8 h after an uninhibited infection; lane 6, mRNA isolated from uninfected Ltkcells; lane 7, mRNA isolated fr-om Ltk cells infected with a tk- mutant of VV (tk-11) 2 h after an uninhibited infection. The migration positions and molecular weights (x10-3) of four brome mosaic virus proteins which were used as molecular weight markers are indicated on the right. cells 5 h after infection in the presence of hydroxyurea also directed the synthesis of very similar products (Fig. 3, lane 4). Such mRNA contained both immediate early and delayed early transcripts. There were minor differences between the products whose syntheses were directed by these miRNA preparations, but no major polypeptides that might have corresponded to the products of delayed early genes were evident. This result was in accordance with the analysis of the polypeptides made in VVinfected cells under these various conditions; no viral polypeptides have been assigned to the delayed early class yet. Late VV mRNA, which was isolated from cells 8 h after infection, was translated with only 20 to 40% the efficiency of early mRNA, even at concentrations which sat-

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urated the cell-free system (see below). As shown in Fig. 3, lane 5, late mRNA directed the synthesis of a series of polypeptides that were quite different and much less complex than the polypeptides whose syntheses were directed by early mRNA. Some of these polypeptides comigrated with the polypeptides synthesized in infected cells 8 h after infection (data not shown). mRNA that was isolated from cells infected with a mutant of VV which lacked a functional tk gene was also used to direct cell-free protein synthesis. The polypeptide products (Fig. 3, lane 7) closely resembled the polypeptides encoded by wild-type mRNA isolated under the same conditions (lane 2). We found no differences in the 40,000- to 42,000-dalton region that could be correlated with the mutant phenotype. Cell-free enzyme synthesis. Recently, we described the cell-free synthesis of active VV thymidine kinase (Hruby and Ball, Virology, in press). Viral mRNA preparations were translated in reticulocyte lysates, and the translation products were assayed for thymidine kinase activity. We used this assay to examine the relative concentrations of functional tk message in VVinfected cells. mRNA isolated from cells 2 h after infection directed the cell-free synthesis of VV thymidine kinase (Fig. 4a and Table 1), as described before (D. E. Hruby and L. A. Ball, Virology, in press). The amount of enzyme synthesized was proportional to the mRNA concentration up to about 65 ,ug/ml, but at higher mRNA concentrations the system approached saturation. Thernal denaturation of the mRNA immediately before translation improved its ability to direct thymidine kinase synthesis by 30 to 50%. On the other hand, late mRNA, which was isolated from cells 8 h after infection, did not direct the synthesis of detectable levels of active thymidine kinase at any mRNA concentration tested, with or without thermal denaturation (Fig. 4c and Table 1). To examine the possibility that a component of the late mRNA preparation inhibited or obscured the translation of tk mRNA, early and late VV mRNA preparations were mixed in various proportions and assayed for the ability to direct cell-free protein and enzyme syntheses. Figure 5 shows no significant deviations from linearity, and thus there was no evidence for an inhibitor of tk mRNA expression in the late VV mRNA preparation. The situation appeared to be straightforward; mRNA extracted from cells that were synthesizing viral thymidine kinase contained functional tk mRNA, whereas mRNA extracted from cells that had stopped synthesizing the enzyme contained no such mRNA activity.

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FIG. 4. Cell-free synthesis of thymidine kinase in response to VV mRNA preparations. Reticulocyte lysates were programmed with different concentrations of mRNA, incubated at 30°C for 4 h, and then assayed for thymidine kinase activity. Two parallel experiments were performed; the VV mRNA was (a) or was not (0) heat denatured immediately before translation. (a) mRNA isolated from Ltk- cells 2 h after an uninhibited infection with wild-type VV. (b) mRNA isolated from Ltk- cells 5 h after an infection with wild-type VV carried out in the presence of hydroxyurea. (c) mRNA isolated from Ltk- cells 8 h after an uninhibited infection with wild-type VV.

Next, we examined whether the disappearance of functional tk nRNA was the cause of the repression of enzyme synthesis 4 h after infection. The substantial tk mRNA activity in message preparations that were isolated from cells 5 h after an infection in the presence of hydroxyurea suggested that this was not the case. At equivalent subsaturating concentrations of total mRNA and at saturation, this mRNA

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TABLE 1. Properties of VV mRNA preparationsa Relative

Relative

Time postinthymifection thionine dine ki(h) incorpo- nase acration tivity 1.0 1.0 2 97.4 48.5 8 38.3 1.0 5 82.5 64.2

[3S]me-

Source of mRNA

None VV-infected cells

VV-infected, hydroxyureatreated cells 5 VV-infected, cycloheximide192.8 237.2 treated cells a [3S]methionine incorporation and thymidine kinase activity were assayed after 2 and 4 h of cell-free protein synthesis, respectively. The results are expressed relative to the endogenous [nS]methionine incorporation and thymidine kinase activity of the reticulocyte lysate, which were 680 cpm of 3S and 395 cpm of 3H, respectively. Each of the mRNA preparations was added at a concentration of 130 pg/ml.

a 40K30

F20-

the former situation, but was maximal under the latter circumstances (Fig. 1). An examination of the complete time course of tk mRNA accumulation and disappearance during a normal, uninhibited VV infection led to the same conclusion. tk mRNA accumulated early and reached peak concentrations 3 to 4 h after infection (Fig. 6b). Thereafter, it disappeared with a half-life of about 1 h, so that by 8 h after infection this activity was barely detectable, as described above (Fig. 4c). A comparison of these measurements with measurements of thymidine kinase synthesis in infected cells showed that substantial levels of functional tk mRNA persisted in cells beyond the time when the synthesis of active enzyme had ceased. For example, at 1.5 and 5 h after infection VV-infected cells contained similar amounts of functional tk mRNA, as assayed by cell-free translation (Fig. 6b). However, at the early time, the cells from which this mRNA was extracted were synthesizing thymidine kinase at maximal rates, whereas at the later time enzyme synthesis was totally repressed (Fig. 1). Therefore, the disappearance of functional mRNA cannot be the basis of the mechanism of tk gene repression in VV-infected L-cells. a

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FIG. 5. Thymidine kinase synthesis (a) andprotein synthesis (b) of reticulocyte lysates programmed with mixtures of mRNA isolated from Ltk- cells 2 h (early) and 8 h (late) after infection with wild-type VV. [3S]methionine incorporation and thymidine kinase activity were measured after 2 and 4 h of incubation, respectively. The total mRNA concentration in each mixture was 65 ltg/ml, which was subsaturating for both protein synthesis and^enzyme synthesis.

directed the synthesis of about 1.5 times more enzyme than the mRNA isolated 2 h after an uninhibited infection (Fig. 4b and Table 1). Nevertheless, in the infected cells from which the mRNA preparations were isolated, the synthesis of thymidine kinase was repressed severely in

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FIG. 6. Protein synthesis (a) and thymidine kinase synthesis (b) of reticulocyte lysates programmed with mRNA isolated from Ltk- cells at different times after an uninhibited infection with wild-type VV. The mRNA concentration in each case was 65 lg/ml. [3S]methionine incorporation and thymidine kinase activity were measured after 2 and 4 h of incubation, respectively.

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The concentration of tk mRNA in cells 5 h after an infection in the presence of hydroxyurea was approximately the same as the peak concentration achieved 3 h after an uninhibited infection. This suggested that in the presence of hydroxyurea, tk mRNA did not continue to accumulate beyond its normal peak concentration. To examine the possibility that the continued accumulation of tk mRNA was prevented by one or more of the early gene products, VV mRNA was isolated 5 h after an infection in the presence of cycloheximide, a drug that restricts the virus to transcription of only the immediate early genes. This mRNA preparation was unusually active in directing the cell-free synthesis both of viral polypeptides (Fig. 3, lane 3) and of thymidine kinase (Table 1). Indeed, the concentration of tk mRNA in this preparation was three- to fourfold higher than the peak concentration achieved during normal infections. These results supported the hypothesis that one or more early gene products are responsible for preventing the continued accumulation of tk mRNA. DISCUSSION The tk gene of VV has been studied widely as a model for the control of viral gene expression (13, 14, 17-21). In a series of elegant experiments performed about 18 years ago, McAuslan showed that tk gene repression was mediated by a late viral gene function which operated at a post-transcriptional level (19, 20). Lacking an assay for tk mRNA, McAuslan was not able to distinguish between repression mechanisms that involved the inactivation or degradation of the mRNA and repression mechanisms that involved a change in the selectivity of the translational machinery of the infected cells. The results described above demonstrate that functional tk mRNA does indeed disappear at late times during VV infection and that this disappearance depends on the expression of late viral genes. However, this does not appear to be the mechanism by which expression of the gene is repressed in L-cells. Active enzyme ceases to accumulate when the cells contain a high concentration of functional mRNA. Moreover, if late viral gene expression is prevented by inhibitors of DNA synthesis, tk mRNA remains at a high level, but it is not translated into active enzyme. We conclude that repression operates at the post-transcriptional level by a mechanism other than irreversible inactivation of tk gene mRNA. mRNA isolated from VV-infected cells at late times after infection contains significant amounts of double-stranded RNA (3). Boone et al. (la) have shown that some of this double-

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stranded RNA results from intermolecular duplex formation between mRNA sequences made early in infection and polyadenylylated "antiearly" sequences that accumulate later. The function of these complementary species is not clear, but their existence raises the possibility that a specific "anti-tk mRNA" sequence is responsible for inhibiting tk mRNA translation late in infection. In this regard, it is interesting that thermal denaturation of early and late VV mRNA preparations immediately before translation increased their message activities significantly. In our experience, this effect is unique to VV mRNA. However, denaturation did not reveal any tk mRNA activity in late VV mRNA preparations, and late mRNA did not inhibit either [3S]methionine incorporation or thymidine kinase synthesis directed by early mRNA. Thus, these results provided no evidence for the participation of "anti-mRNA" sequences in the repression of thymidine kinase synthesis. In VVinfected L-cells, unlike HeLa cells, this repression is a function of an immediate early or delayed early viral gene, but details concerning the gene(s) involved and the precise mechanism must await further study. Superimposed on this control mechanism, which regulates the amount of active enzyme that accumulates, two other controls which regulate the amount of functional tk mRNA can be discerned. Inhibitors of protein synthesis stimulate and prolong transcription by viral cores during the initial stages of infection. This has been shown for the unresolved products of the immediate early genes (7) and was confirmed specifically for the tk gene by our results. A probable explanation of this effect is that one or more of the immediate early gene products inhibits early transcription, including transcription of the tk gene. For example, in the presence of hydroxyurea tk mRNA accumulates, but only to its normal peak concentration, whereas cycloheximide causes this level to be exceeded by three- to fourfold. The relationship between this inhibition of early transcription and the second stage of viral uncoating, which is also mediated by an immediate early gene product, remains to be determined. A late gene function is responsible for the disappearance of tk mRNA from the cytoplasm of infected cells at later times after infection. It is possible that in some cells (e.g., HeLa cells) this loss of functional tk mRNA may be the dominant mechanism that limits accumulation of the enzyme. This suggestion would reconcile the results of McAuslan (19, 20) and Jungwirth and Joklik (13) with our data and those of Zaslavsky and Yacobson (31). An alternative possi-

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bility is that the same regulatory functions may be expressed in different temporal classes in different poxvirus-cell systems. It remains to be determined whether the short functional half-life of the tk message is representative of all mRNA's 4 h after infection or whether the mechanisms responsible for mRNA inactivation are selective. The complex patterns of polypeptide synthesis and repression suggest that some selectivity probably exists, but hybridization studies have indicated that the rate of degradation of total VV mRNA increases markedly during infection (26). In any case, the elucidation of the mechanisms of translational control and inactivation of VV tk mRNA should provide useful information concerning the regulation of gene expression in this system. ACKNOWLEDGMENTS This work was supported by Public Health Service Fellowship AI 05892 from the National Institutes of Health to D.E.H. and by Public Health Service grant AI 16356 and Public Health Service Research Career Development Award AI 00378 from the National Institutes of Health to L.A.B. LITERATURE CITED 1. Belle Isle, H., S. Venkatesan, and B. Moss. 1981. Cellfree translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome. Virology 112:306-317. la.Boone, R. F., R. P. Parr, and B. Moss. 1979. Intermolecular duplexes formed from polyadenylated vaccinia virus RNA. J. Virol. 30:365-374. 2. Chipchase, M., F. Schwendimann, and R. Wyler. 1980. A map of the late proteins of vaccinia virus. Virology 105:261-264. 3. Colby, C., C. Jurale, and J. R. Kates. 1971. Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro. J. Virol. 7:71-76. 4. Cooper, J. A., and B. Moss. 1979. In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells. Virology 96:368-380. 5. Cremer, K., M. Bodemer, and W. C. Summers. 1978. Characterization of the mRNA for herpes simplex virus thymidine kinase by cell-free synthesis of active enzyme. Nucleic Acids Res. 5:2333-2344. 6. Dubbs, D. R., and S. Kit. 1964. Isolation and properties of vaccinia mutants deficient in thymidine kinase-inducing activity. Virology 22:214-225. 7. Esteban, M., and D. H. Metz. 1973. Early virus protein synthesis in vaccinia virus-infected cells. J. Gen. Virol. 19:201-216. 8. Geshelin, P., and K. I. Berns. 1974. Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. 88:785-796. 9. Glisin, V., R. Crkuenjakov, and C. Byus. 1974. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633-2637. 10. Hruby, D. E., L. A. Guarino, and J. R. Kates. 1979. Vaccinia virus replication. I. Requirement for the host cell nucleus. J. Virol. 29:705-715. 11. Hruby, D. E., D. L. Lynn, and J. R. Kates. 1979. Vaccinia virus replication requires active participation of the host cell transcriptional apparatus. Proc. Natl.

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