Replication of vaccinia DNA in mouse L cells IV. Protein synthesis and viral DNA replication

VIROLOGY

86,376-390

(1978)

Replication

of Vaccinia DNA in Mouse L Cells

IV. Protein Synthesis MARIANO

ESTEBAN

and Viral DNA Replication AND

JOHN

A. HOLOWCZAKl

Department of Microbiology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854 Accepted December 16, 1977 The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 ag/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [“Hlthymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, p = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, p = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes. INTRODUCTION

Inhibitors of protein synthesis, when present during the period of DNA synthesis, have been found to prevent the normal continuation of DNA replication in many types of eukaryotic cells (Mueller et al., 1962; Bennet et al., 1964; Littlefield and Jacobs, 1965; Taylor, 1965; Cummins and Rusch, 1966; Young, 1966; Kim et al., 1968; Brown et aZ., 1970; Highfield and Dewey, 1972). Some controversy exists as to whether the observed inhibition of DNA synthesis is due to a reduction in the initiation of synthesis of the many replication units comprising the eukaryotic genome or to an inhibition of chain elongation within ’ To whom dressed.

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replication units (Weintraub and Holtzer, 1972; Gautschi and Kern, 1973; Hand and Tamm, 1973; Hori and Lark, 1973; Gautschi, 1974; Housman and Huberman, 1975; Searle and Simpson, 1975; Evans et al., 1976; Funderud and Havgli, 1977). In prokaryotic cells new proteins are needed only for the initiation of DNA replication (Lark, 1969,1972; Kornberg, 1974). Protein synthesis has also been shown to be required for viral DNA replication after infection of animal cells. In the case of the papovaviruses, inhibition of protein synthesis resulted in a decrease in the rate of viral DNA synthesis, and it has been suggested that protein synthesis is required to initiate DNA synthesis (Branton et al., 1970; Branton and Sheinin, 1968, 1973; Kit and Nakajima, 1971; Kang et al., 1971; Bourgaux 376

0042~6822/78/0862-0376$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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and Bourgaux-Ramoisy, 1972; Yu et al., 1975). With adenoviruses, the initiation of viral DNA synthesis was insensitive to’inhibitors of protein synthesis when such inhibitors were added shortly after the appearance of progeny viral DNA (Horwitz et al., 1973). With herpesviruses (Roizman and Roane, 1964; Kaplan and Ben-Porat, 1966; Ben-Porat and Kaplan, 1973), continuous protein synthesis was shown to be required for viral DNA synthesis, but whether the initiation of DNA replication or chain elongation was affected was not examined. Addition of inhibitors of protein synthesis to poxvirus-infected cells after uncoating is completed and “early” proteins are synthesized causes viral DNA synthesis to be blocked (Kates and McAuslan, 1967; Bedson and Cruikshank, 1968). The protein requirement for viral DNA replication appears to differ from the known poxvirusinduced enzymes that might participate in DNA replication in terms of the stability of its messenger and its apparent noncatalytic relation to DNA synthesis (Kates and McAuslan, 1967). The step in DNA replication which is affected has not been examined. When DNA replication has been completed or is near completion, continuous protein synthesis is required for the release of replicated vaccinia DNA from cytoplasmic DNA-protein complexes (Dahl and Kates, 1970; Esteban and Holowczak, 1977b). It has been suggested that polypeptides required for the initial phase of viral assembly must be synthesized to allow release of newly replicated DNA from DNA-protein complexes (Dahl and Kates, 1970). In this report, the effect of cycloheximide, a potent inhibitor of protein synthesis in mammalian cells (Columbo et al., 1966) on the replication of vaccinia DNA in mouse L cells was investigated. Analysis of viral DNA replication showed that under conditions where “early” viral functions are expressed, an initial round of viral DNA replication can occur in the absence of protein synthesis. In order for additional rounds of viral DNA replication to be initiated, new protein synthesis was required. The release of newly replicated viral DNA from cyto-

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plasmic complexes was inhibited by cycloheximide but not rifampin. This suggests that the proteins required for release of the progeny DNA molecules from complexes are distinct from those proteins required to initiate virion assembly. MATERIALS

AND

METHODS

Cells, virus, and methods for analysis of viral DNA. Propagation of mouse L cells and purification of virus was carried out as described previously (Esteban and Holowczak, 1977a, 197713). Unless otherwise noted, mouse L cells were infected at a multiplicity of 1000 EB/cell. Techniques for analysis of viral DNA by sedimentation in neutral or alkaline sucrose gradients as well as density equilibrium centrifugation in neutral CsCl have been described in detail elsewhere (Esteban and Holowczak, 1977a). Hydroxyurea (HU) was employed to inhibit viral DNA replication. The effects of this drug on host and viral macromolecular synthesis and methods for reversing its activity have been reported (Rose&ram et al., 1966; Pogo and Dales, 1969, 1971; Fil et al., 1974). Fractionation of infected cell lysates by sedimentation in neutral sucrose gradients to yield DNA-protein complexes containing newly replicated viral DNA as a “pellet fraction,” while “released” viral DNA is resolved in the gradients, was performed as described previously (Esteban and Holowczak, 1977b). Chemicals and radioisotopes. Hydroxyurea, cycloheximide (Acti-Dione), and rifampin were purchased from Mann Research Laboratories, New York. The [methyZ-3H]thymidine (2.20 Ci/mmol and [2-‘4CJthymidine (25 mCi/mmol) were purchased from the New England Nuclear Corp. Sarkosyl NL-97 was obtained from Giegy Chemical Co. RESULTS

(I) The Effect Formation DNA

of Cycloheximide on the of Crosslinked Vaccinia

When infected at a multiplicity of 1000 EB/cell, viral DNA synthesis in mouse L cells begins at about 1 hr p.i., reaches a peak between 2 and 3 hr p.i., and then declines by 4 hr p.i. (Esteban and Holow-

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czak, 1977a). As shown in Fig. 1, during continuous labeling, the rate of incorporation of [3H]thymidine into acid-precipitable radioactivity in intact virus-infected cells is several times greater than that measured in uninfected cells. Addition of cycloheximide to the infected cells causes a marked decrease in the incorporation of [3H]thymidine into acid-precipitable material (Fig. 1). Similar results have been reported when rabbitpox DNA replication was studied and either puromycin or cycloheximide was added to inhibit protein synthesis shortly after the onset of viral DNA synthesis (Kates and McAuslan, 1967). The effect of cycloheximide on the synthesis of vaccinia DNA was investigated using techniques described previously (Esteban and Holowczak, 1977a). Infected cells were pulse labeled for 15 min with [3H]thymidine at 1.5, 2, 2.5, 3, 3.5, and 4 hr p.i. After the 15min labeling period, cycloheximide (100 pg/ml) was added to inhibit protein synthesis and the label was chased with 2 mM cold thymidine. At 7 hr p.i., 2 X lo5 cells were harvested from each culture and mixed with “alkaline” lysis solution on the surface

of alkaline sucrose gradients. The cells were allowed to lyse (18 hr, 4”, in the dark), the gradients were centrifuged, hactionated, and the distribution of the radioactivity in such gradients was determined (Fig. 2) (Esteban and Holowczak, 1977a). In vaccinia virus-infected cells not treated with cycloheximide, viral DNA molecules synthesized during a 15-min pulse 2 hr p.i. sedimented with a peak at about 40 S, relative to adenovirus type 2 DNA; by 7 hr p.i., over 70% of the label had been chased into molecules which sedimented at 102 to 106 S (Fig. 2G). Viral genomes, having two crosslinks, would be expected to sediment at 102 to 106 S in alkaline sucrose gradients (Geshelin and Berns, 1974; Holowczak, 1976; Pogo, 1977; Esteban and Holowczak, 1977a). In cycloheximide-treated infected cells, the short DNA strands labeled during a 15-min pulse with r3H]thymidine could be chased into molecules which co-sedimented with viral DNA having one crosslink (92-94 S) or two crosslinks (102-106 S) (Fig. 2, panels A-F) (Esteban and Holowczak, 1977a). The results of these experiments showed that replication of vaccinia DNA in the presence of cycloheximide proceeded in a fashion similar to that in infected cells where protein synthesis was not inhibited. Since mature crosslinked molecules were completed, the pulse-labeled fragments synthesized prior to the addition of cycloheximide continued to be elongated into mature viral DNA. Moreover, the rate of chain elongation was comparable to that measured in cells synthesizing protein continuously after infection (Fig. 2) (Esteban and Holowczak, 1977a).

FIG. 1. Kinetics of DNA synthesis in untreated and cycloheximide-treated cells. Infected or mock-infected cell cultures were prepared as described under Materials and Methods. At the end of the adsorption period one-half of the infected or mock-infected cells were diluted into medium containing cycloheximide (100 pg/ml). The cultures were labeled with 2 pCi/ml of [SHlthymidine beginning 2 hr p.i. At the times indicated after virus infection, 1 x IO” cells were removed from the cultures, collected by centrifugation, and washed two times in buffered saline, and the radioactivity incorporated into TCA-precipitable material was determined. (0) Infected cells; (A) cycloheximidetreated infected cells; (0) mock-infected cells; (A) cycloheximide-treated mock-infected cells.

(2) The Effect of Cycloheximide on the Initiation and Elongation of Replicating Viral DNA To clarify further the effect that inhibition of protein synthesis has on vaccinia DNA replication, the following experiments were performed. Cells were infected in the presence of hydroxyurea (5 n&f), a reversible inhibitor of poxvirus DNA replication (Pogo and Dales, 1969; Rosenkranz et al., 1966; Fil et al., 1974). In the presence of inhibitors of viral DNA replication, early mRNA sequences are transcribed and

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FIG. 2. Effect of cycloheximide on the formation of cross-linked vaccinia DNA. Infected or mock-infected cells were prepared as described under Materials and Methods. As the times indicated after virus infection, 5 x ld cells were removed from the cultures and pulse labeled with 10 &i/ml of [“Hlthymidine for 15 min. The label was then chased with cold thymidine (2 m&f) in the presence or absence of cycloheximide. At 7 hr p.i., 1 x 10’ cells were removed from the cultures and washed in buffered saline, and 2 X lo” cells were loaded on a 15 to 30% alkaline sucrose gradient overlaid with an 0.8-ml layer of lysis solution. Lysis of the cells and conditions for sedimentation analysis were as described under Materials and Methods. (A) Cells pulse-labeled for 15 min at 1.5 hr pi. and then chased up to 7 hr p.i. (B) Cells pulse-labeled for 15 min at 2 hr pi. and then chased up to 7 hr p.i. (C) Cells pulse-labeled for 15 min at 2.5 hr p.i. and then chased up to 7 hr p.i. (D) Cells pulse-labeled for 15 min at 3.0 hr p.i. and then chased for 7 hr pi: (E) Cells pulse-labeled for 15 min at 3.5 hr p.i. and then chased up to 7 hr p.i. (F) Cells pulse-labeled for 15 min at 4 hr p.i. and then chased up to 7 hr p.i. Samples A through F were chased in the presence of cycloheximide. (a), infected cells; (A), uninfected cells. Gradients in A through F were centrifuged at 39,000 rpm for 1 hr.

translated into viral specific proteins, in&ding those required for viral DNA replication (reviewed in Moss, 1974). At 3.75 hr p.i., the infected and mock-infected cultures were divided, and one-half of each

culture was treated with cycloheximide (100 pg/ml). At 4 hr p.i., hydroxyurea was removed from the cells by repeated washes (Pongo and Dales, 1971) with complete medium. Cycloheximide was present in the

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FIG. 2. Continued. In (G) samples were centrifuged at 39,090 rpm for 3 hr. (0) Infected cells pulse-labeled for 15 min at 2 hr p.i. and then immediately lysed on top of the gradient; (0) infected cells pulse-labeled for 15 min at 2 hr p.i. and chased up to 7 hr pi. and then analyzed; (A) mock-infected cells pulse-labeled for 15 min at 2 hr pi. and then chased up to 7 hr p.i.

medium used to wash the cells when appropriate. The cells were then diluted into prewarmed, complete medium with or without cycloheximide (Time zero) and pulselabeled with r3H]thymidine at different times after HU reversal. The kinetics of incorporation of [3H]thymidine during lo-min pulses into acid-precipitable radioactivity in infected and mock-infected cells with or without cycloheximide-treatment is indicated in Fig. 3. The rate of viral DNA synthesis in both untreated and cycloheximide-treated, infected cells was the same during the first 30 min after HU reversal. Thereafter, the rate of r3H]thymidine incorporation in cycloheximide-treated cells dropped sharply, and by 2 hr post-HU reversal incorporation was suppressed by greater than 90%. Sedimentation analysis in alkaline sucrose gradients showed that after HU reversal the majority of viral DNA molecules labeled during a 2-min pulse in cycloheximide-treated cells sedimented at about 10 to 30 S (Fig. 4). With prolonged pulses of 15 and 30 min, the size of the replicating DNA molecules increased and DNA species sedimenting at about 20 to 40 S and 30 to 50 S were observed. Full-length viral DNA molecules sedimenting at 70 to 72 S were detected after a 30-min pulse. Molecules which sedimented at 92 to 94 S and 102 to

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FIG. 3. Effect of cycloheximide on the kinetics of DNA synthesis after reversal of a hydroxyurea (HU) block. Infected or mock-infected cells were prepared as described under Materials and Methods. At the end of the adsorption period (15 min), cells were diluted into medium containing 5 mM HU. At 3.75 hr p.i., half of the cultures were treated with cycloheximide. At 4 hr p.i., the HU block was reversed as described under Materials and Methods. Cells were washed with complete medium (plus or minus cycloheximide) cooled to 4” to minimize the initiation of viral DNA replication during the washes. After washing, infected or mockinfected cells were resuspended in warmed (37”) fresh medium (plus or minus cycloheximide) at a concentration of 1 X 10” cells/ml. At the times indicated after HU reversal, 1 X ld cells were removed from the cultures and pulse labeled for 10 min with [“Hlthymidine. The determination of TCA-precipitable radioactivity was as indicated in Fig. 1. (0) infected cells; (A) cycloheximide-treated infected cells; (0) mockmock-ininfected cells; (A) cycloheximide-treated fected cells.

106 S were labeled significantly only after a 60-min pulse (Fig. 4). The results of these experiments demonstrated that in cycloheximide-treated vaccinia virus-infected cells, the distribution of radioactivity in viral DNA molecules synthesized during short pulses or after long labeling times (Figs. 2 and 4) was the same as that detected in untreated infected cells (Esteban and Holowczak, 1977a). The formation of “intermediate strands” and, finally, crosslinked mature viral DNA molecules was not affected when protein synthesis was inhibited. At the concentrations of cycloheximide employed in these studies, protein synthesis was inhibited by greater than 90% 15 min after addition of the drug (data not shown).

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As shown above, neither the initiation nor the elongation of the viral DNA molecules synthesized during the first 30 min after HU reversal was affected by cycloheximide. The incorporation of C3H]thymidine into acid-insoluble material was, however, drastically reduced after this initial 30-min period, suggesting that new rounds of viral DNA replication may be prevented when protein synthesis is inhibited. To test this possibility, infected cells after HU reversal, with or without cycloheximide treatment, were pulse labeled for 30 min with r3H]thymidine and then chased with 100 pg/ml of BuDR containing 1 pg/ml of FudR and 10 pg/ml of uridine. The results of such an experiment are presented in Fig. 5. Viral DNA molecules synthesized in cells after HU reversal and not treated with cycloheximide have their density shifted during the chase with BuDR from 11 (p = 1.712 g/ml) to hl (p = 1.77 g/ml) and finally to hh (p = 1.825 g/ml). We have shown previously that the density of 11 molecules which differs from that of DNA extracted from vii-ions is due to the presence of significant ssDNA regions in such replicating molecules (Esteban and Holowczak, 1977a). In cycloheximide-treated infected cells, the majority of the viral DNA molecules have their density shifted from 11to hl during the BuDR chase. The results showed that during the semiconservative synthesis of vaccinia DNA (Esteban and Holowczak, 1977a), the conversion of hybrid DNA molecules to hh viral DNA molecules did not occur in cycloheximide-treated cells. This demonstrated that initiation of new rounds of viral DNA replication is inhibited when cycloheximide is present. In addition, repair synthesis of viral DNA in the presence of cycloheximide must be very limited or does not occur, since the majority of the labeled viral DNA molecules synthesized after treatment with the drug were converted to hybrid molecules. (3) The Effect of Cycloheximide on the Formation of Viral Factories and Release of Newly Replicated Genomes from Factories Poxvirus DNA in association with certain proteins replicates in large cytoplasmic in-

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elusions or “factories (reviewed in Moss, I974).” By using a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral factories in vaccinia virus-infected cells can be determined (Esteban, 1977). The effect of cycloheximide on the formation of the viral factories was studied after HU reversal. As can be observed in Fig. 6C, the viral factories formed in cycloheximidetreated cells 4 hr after HU-reversal were similar in appearance but less abundant than those seen in untreated infected cells (Fig. 6B). No viral factories were observed in uninfected cells (Fig. 6A) or in infected cells maintained in the presence of HU (Fig. 6D). At 18 hr p.i., the size and morphology of the factories in cycloheximide-treated infected cells (Fig. 6F) were similar to those observed 4 hr post-HU reversal In infected cells continuously synthesizing protein (Fig. 6E), the viral factories increased in size with time after HU reversal, forming large masses of granular cytoplasmic inclusions around the highly fluorescent nuclei. The results of the experiments described above demonstrated that viral factories are formed in cycloheximide-treated infected cells, reflecting the fact that the initial round of DNA replication is not affected when protein synthesis is inhibited. However, viral factories are reduced in number and do not increase in size with time after infection in cycloheximde-treated cells, indicating that viral DNA replication is interrupted. Analysis of the data presented in Fig. 4 showed that the specific activity of the synthesized viral DNA in both untreated and cycloheximide-treated infected cells during the first 30 min after HU reversal was the same. We conclude that the inhibition of incorporation of r3H]thymidine into TCA-precipitable material in cycloheximide-treated cells (Figs. 1 and 3) reflects primarily an inhibition of viral DNA synthesis rather than a change in the cellular TTP pool size. It has been previously shown that the vaccinia factories are large viral DNA-protein complexes from which “intermediate” DNA strands and mature viral DNA molecules are gradually released as vaccinia DNA replication progresses and

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the assembly of virions begins (Joklik and Becker, 1964; Esteban and Holowczak, 1977a;Esteban and Holowczak, 1977b). The effect of cycloheximide on the release of viral DNA molecules synthesized after HU reversal from DNA complexes was investigated. Inhibition of protein synthesis with cycloheximide could affect the viral factories in the following ways: (i) by inhibiting the synthesis of protein(s) with affinity for the viral DNA which may be required for the stability of the complexes; (ii) by inhibiting the synthesis of protein(s) required for the release of mature viral DNA from the complexes as a step necessary for virion assembly; (iii) by causing premature detachment of the nascent chains from their DNA templates during replication. The last possibility is unlikely, as no accumulation of short strands of DNA was observed during different labeling times (see Figs. 2 and 4) after addition of cycloheximide. We have tested directly the other two possibilities by sedimentation analysis in neutral sucrose gradients of the newly replicated viral DNA. Under the experimental conditions used, intermediate strands and mature viral DNA molecules which are released from the DNA complexes can be resolved in the gradients, while viral DNA associated with the large complexes pellets to the bottom of the centrifuge tube with cellular DNA (Esteban and Holowczak, 1977a, 197713). During the period that the infected cells are treated with HU, host macromolecular synthesis is inhibited (Pogo and Dales, 1971) and after reversal of the HU block, host DNA is not significantly labeled. Infected cells after HU reversal, with or with-

HOLOWCZAK

out cycloheximide treatment, were labeled with r3H]thymidine for 30 min and then chased with 2 mM cold thymidine. At 1, 2, and 3 hr after HU reversal, 1 x lo6 cells were removed from the cultures and washed, and 2 X ld cells were mixed with neutral lysis buffer on the surface of neutral sucrose gradients. When lysis was complete (18 hr, 4”, in the dark), the gradients were centrifuged, fractionated, and the distribution of radioactivity in the gradients was determined as previously described (Esteban and Holowczak, 1977a). The results of these analyses are presented in Fig. 7. In virus-infected cells where protein synthesis was not inhibited after HU reversal intermediate DNA strands (sedimenting slower than 70-72 S) and mature viral DNA molecules (sedimenting at 68-72 S) are gradually released from complexes. By 3 hr after HU reversal, about 60% of the labeled viral DNA could be released from complexes under our conditions of analysis and cosedimented with mature viral DNA. In contrast, in cycloheximide-treated infected cells, over 90% of the newly replicated labeled viral DNA remained associated with the DNA-protein complexes at all tme intervals examined after HU reversal. These results confirmed and extended our previous observations (Esteban and Holowczak, 1977b) and showed that continuous protein synthesis and presumably expression of a “late” viral function are required for the release of viral DNA synthesized after HU reversal from the complexes or factories. It has been suggested that the protein required for the release of newly replicated

FIG. 4. Sedimentation analysis in alkaline sucrose gradients of replicating viral DNA synthesized after HU reversal in the presence or absence of cycloheximide. Infected or mock-infected cells were treated with HU as described in Fig. 3 and under Materials and Methods. At 3.75 hr pi., half of the cultures were treated with cycloheximide, the HU block was reversed at 4 hr p.i., and the cells were resuspended in fresh medium (1 x 10” cells/ml) with or without cycloheximide. After HU reversal, cells were labeled with 10 pCi/ml of [“Hlthymidine (A, B, C) and 1 &i/ml of [‘?]thymidine (D, E, F). At the times indicated, LO-ml aliquots were removed from the cultures and washed in buffered saline, and 2 X IO” cells were loaded on a 15 to 30% alkaline sucrose gradient overlaid with an 0.8-ml layer of lysis solution. Lysis of the cells and conditions for sedimentation analysis were as described under Materials and Methods. (A) Cells pulse-labeled for 2 min. (B) Cells pulse-labeled for 15 min. (C) Cells pulse-labeled for 30 min. (D) Cells pulse-labeled for 1 hr. (E) Cells pulse-labeled for 2 hr. (F) Cells pulse-labeled for 3 hr. (0) Infected cells; (0) cycloheximide-treated infected cells. The percentages of total [‘?]thymidine-labeled DNA recovered in the pellet fraction of the gradients were, respectively, D, 9% (0) and 8% (0); E, 14% (0) and 17% (0); F, 16% (0) and 17% (0); less than 600 “H cpm were incorporated into cellular DNA during a 30-min labeling period, and about 100 14Ccpm were incorporated during a 3-hr labeling period.

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viral genomes from complexes may be involved in the initial stages of virion assembly (Dahl and Kates, 1970). When infected cells were treated with rifampin after HU reversal, the assembly of virions was blocked (Fig. 8A) but the release of genomes from complexes was not affected (Fig. BB). The synthesis of viral DNA molecules (Esteban, 1977) and, as demonstrated by the experiments described above, the release of newly replicated genomes from complexes can occur in the absence of viral assembly. DISCUSSION

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FIG. 5. Equilibrium density centrifugation in neutral CsCl of replicating viral DNA synthesized after HU reversal in the presence or absence of cycloheximide. Infected and mock-infected cells were prepared and treated with HU as described in Fig. 3 and under Materials and Methods. At 3.75 hr p.i., half of the cultures were treated with cycloheximide, the HU block was reversed at 4 hr p.i., and cultures were resuspended in fresh medium (1 x IO6 cells/ml) with or without cycloheximide. After HU reversal, cells were pulse-labeled with 10 @/ml of [“Hlthymidine for 30 min and then chased in medium containing BUdR (100 pg/ml), FUdR (1 pg/ml), and uridine (10 pg/ml). At the indicated times, 1 x ld cells were removed from the cultures, collected by centrifugation, washed two times in buffered saline, resuspended in 0.4 ml of buffered saline, and dissolved in 1% Sarkosyl in the presence of 30 mM BME. After incubation for 10 min at room temperature, the lysate was digested with 1 mg/ml of pronase for 2 hr at 37”. The pronasetreated lysate from 1 X 19’ cells was analyzed by equilibrium density centrifugation in neutral CsCl as described under Materials and Methods. (A) Infected cells labeled for 30 min and then chased for up to 1 hr. (B) Infected cells labeled for 30 min and chased for up

Two events related to the replication of vaccinia virus DNA are sensitive to the presence of inhibitors of protein synthesis. First, experimental evidence has been provided here to show that after an initial round of DNA replication has been completed, each new round of viral DNA replication must be accompanied by the synthesis of protein. Second, we have confirmed and extended earlier observations (Dahl and Kates, 1970; Esteban and Holowczak, 1977b) concerning the requirement for continuous protein synthesis in order for newly replicated viral genomes to be released from DNA-protein complexes or factories. When cycloheximide was added to infected cells after the start of viral DNA synthesis, those DNA molecules whose replication was initiated were completed and mature, and crosslinked molecules accumulated (Fig. 2). Subsequently, DNA replication in the absence of new protein synthesis was inhibited (Fig. 1). To study this effect in more detail, cells were treated with hydroxyurea to allow expression of early viral functions; cycloheximide then was added to inhibit further protein synthesis, to 2 hr. (C) Infected cells labeled for 30 min and chased for up to 3 hr. (0) Labeled DNA from infected cells treated with cychloheximide and chased with cold BUdR; (0) labeled DNA from infected cells not treated with cycloheximide and chased with cold BUdR. HH, HL, and LL indicate the positions in the gradient where heavy (hh) (p = 1.825 g/ml), hybrid (hl) (p = I.77 g/ml), and light (11) (p = 1.712 g/ml) replicating vaccinia DNA banded at equilibrium (Esteban and Holowczak, 1977a).

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FIG. 6. Fluorescent light micrographs of vaccinia virus-infected cells after HU reversal stained with the bisbenzimide derivative, Hoechst 3358. Infected and mock-infected cells were prepared and treated with HU as described in Fig. 3 and under Materials and Methods. At 3.75 hr pi., half of the cultures were treated with cycloheximide, the HU block was reversed at 4 hr p.i., and cultures were resuspended in fresh medium (1 x 10” cells/ml) in the presence or absence of cycloheximide. Aliquots (1 X 10” cells) were seeded in petri dishes (35 mm) with coverslips and, at appropriate times, stained with the bisbenzimide derivative Hoechst 3358, as previously described (Esteban, 1977) (A) Mock-infected cells; (B) infected cells stained at 4 hr after reversal of HU; (Cl cycloheximide-treated infected cells, stained 4 hr after reversal of HU; (D) infected cells maintained in the presence of HU, stained 4 hr post-infection; (E) infected cells stained at 18 hr after reversal of HU; !F) cycloheximide-treated infected cells, stained at 18 hr after reversal of HU. The arrows (El point to the large cytoplasmic inclusions observed after staining of infected cells. In A only the cell nucleus was stained.

and the hydroxyurea block was reversed to allow viral DNA synthesis to occur (Figs. 3 and 4). Under these conditions, viral DNA

synthesis as measured by the incorporation of [3H]thymidine into acid-insoluble material was identical for 30 min after reversal

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FIG. 7. Neutral sucrose sedimentation analysis of replicating viral DNA synthesized after HU reversal in the presence or absence of cycloheximide. Infected and mock-infected cell cultures were prepared as described in Fig. 3 and under Materials and Methods. After HU reversal, cells were labeled with 10 pCi/ml of [SH]thymidine for 30 min and then chased with 2 m&f cold thymidine. At the indicated times, 1 x IO” cells were removed from the cultures, collected by centrifugation, and washed two times in buffered saline, and 2 X 10’ cells were loaded on a 15 to 30% neutral sucrose gradient overlaid with an 0.8-ml layer of “neutral lysis solution.” Lysis of the cells, centrifugation, and determination of radioactivity were as described under Materials and Methods. (A) Infected cells pulse labeled for 30 min and chased for up to 1 hr. (B) Infected cells pulse labeled for 30 min and chased for up to 2 hr. (C) Infected cells pulse labeled for 30 min and chased for up to 3 hr. (0) Labeled DNA from infected cells; (0) labeled DNA from cycloheximide-treated infected cells. The percentages of total radioactivity recovered in the pellet (complex fraction) of the gradients were, respectively, A, 84% (a) and 91% (0); B, 81% (0) and 93% (0); C, 42% (0) and 91% (0).

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of the HU block in cells continuously synthesizing protein as compared to cells treated with cycloheximide to inhibit protein synthesis. We have previously reported that the replication of full-length vaccinia DNA molecules, which sediment at 70 to 72 S when analyzed in alkaline sucrose gradients, requires about 30 min with a polymerization rate of about 6500 nucleotide pairs/min (Esteban and Holowczak, 1977a). The time period after HU reversal, when DNA replication was similar for cycloheximide-treated or -untreated infected cells, was sufficient to allow the completion of one round of DNA replication. Furthermore, the intermediates detected during this period of viral DNA synthesis and the rate at which chain elongation occurred were similar in cells synthesizing protein as compared to cells treated with cycloheximide (Fig. 4) (Esteban and Holowczak, 1977a). At the levels of cycloheximide employed in these studies, protein synthesis was completely inhibited (XX%) 15 min after addition of the drug. This would indicate that all of the enzymes required for vaccinia DNA replication, including those which introduce crosslinks into the genome, are synthesized early after infection. Since the introduction of crosslinks into newly synthesized DNA is a relatively late event during viral DNA replication (Esteban and Holowczak, 1977a), the enzymes responsible for this process must be stable. We conclude that the protein(s) required for the continuation of viral DNA replication is not one of the poxvirus-induced enzymes which participates in the initial round of DNA replication. The nature of the protein needed for maintaining DNA replication remains to be determined. It has been suggested that histone-like proteins are involved in the control of DNA chain elongation in eukaryotic cells (Weintraub, 1973) and arginine-rich, histone-like polypeptides associated with vaccinia DNA have been detected in the cytoplasm of infected cells (Pogo et al., 1975). In addition, a number of polypeptides with affinity for DNA can be recovered in the form of “virosomes” from infected cells (Polisky and Kates, 1972). Two polypeptides account for 50 to 70% of

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FIG. 8. Effect of rifampin on the assembly of virions and release of newly replicated viral DNA from complexes after HU reversal. (A) Effect of rifampin on assembly of virions after HU reversal. Infected cell cultures containing 2 X 10’ cells were treated as follows: (i) no inhibitors added, w (ii) rifampin (100 pg/ml) added immediately after adsorption, X-X; (iii) HU added at Time 0 and reversed at 4 hr pi., &Q (iv) as in (iii) but after reversal of the HU block, cells were diluted into medium containing rifampin (106 yg/ml), A-A. [JHlThymidine (5 PC/ml) was added to label viral DNA. At 20 hr p.i. (16 hr after HU reversal), the virions were purified and, as shown in A, were sedimented into 20 to 40% sucrose gradients (Holowczak and Joklik, 1967). The position of purified [‘4C]thymidine-labeled virions in the gradients is indicated. (B) Effect of rifampin on the release of viral DNA from complexes. Infected cell cultures prepared as described in Fig. 7, after reversal of a HU block, were treated as follows: (i) diluted into medium containing rifampin (100 pg/ml), H, (ii) diluted (iii) diluted into medium into medium containing rifampin (100 pg/ml) and cycloheximide (100 pg/ml), A-A, containing cycloheximide (100 pg/ml), C-O. Infected cells were pulse labeled with [“Hlthymidine for 30 min post-HU reversal and chased with 2 m&f cold thymidine, and at 6 hr post-HU reversal 2 x 10” cells from each culture were analyzed in neutral sucrose gradients as described in Fig. 7. Radioactivity recovered in pellet (complex fraction) from each gradient was as follows: (i) 39%; (ii) 91%; (iii) 89%. The position of rH]thymidinelabeled DNA released from purified virions and analyzed at the same time in a parallel gradient is indicated.

the proteins found in virosomes, but these polypeptides are present in only trace amounts in assembled virions (Sarov and Joklik, 1973) and could have a function related to viral DNA replication. Finally, we have recently obtained evidence for the presence of protein species relatively resistant to pronase digestion linked to the termini of vaccinia DNA (Soloski, Esteban, and Holowczak, manuscript in preparation). It has been suggested that proteins covalently bound to the termini of adenovirus DNA have an important role in the initiation of adenovirus DNA replication (Rekosh et al., 1977). The proteins we have detected bound to vaccinia DNA could

have a similar function. That the initiation of vaccinia DNA replication, rather than some stage during synthesis, was affected by the inhibition of protein synthesis was established in the following manner. In a number of experiments it was possible to show that DNA molecules whose synthesis was initiated at the time cycloheximide was added were subsequently replicated in the same manner and at the same rate as viral DNA molecules replicating in cells continuously synthesizing protein. Elongation, termination, and crosslinking of replicating viral DNA molecules were not affected when protein synthesis was inhibited. Den-

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sity-shift experiments (Fig. 5) showed that in the presence of cycloheximide after HU reversal, hybrid DNA molecules accumulated. Conversion of hybrid DNA molecules to l&DNA molecules during a chase with BuDR under the conditions described was observed to occur only in cells continuously synthesizing protein. Sedimentation analysis in alkaline sucrose gradients at different times after HU reversal (data not shown) showed that in the presence or absence of protein synthesis, molecules of similar size were labeled during short pulses with [3H]thymidine. The amount of label incorporated into TCA-precipitable molecules was, however, significantly reduced in the cycloheximide-treated cells, reflecting the kinetics of incorporation shown in Fig. 3. The results of these experiments support the conclusion that after an initial round of viral DNA replication has been completed, subsequent rounds of DNA replication are sensitive to inhibitors of protein synthesis. The inhibition of viral DNA synthesis in the presence of cycloheximide, as measured by the incorporation of [3H]thymidine, could be an artifact and result from a perturbation of the TTP pool size, as has been suggested to occur in other systems (Bersier and Braun, 1974; Evans et al., 1976). We argue against such an effect in vacciniainfected cells after HU reversal on the following grounds. First, the specific activity of the labeled viral DNA species detected after analysis in alkaline sucrose gradients during the first 30 min after HU reversal was the same in cycloheximide-treated and untreated cultures (Fig. 4). Second, as an independent measure of DNA synthesis, the morphology and size of factories appearing in the cytoplasm of infected cells was studied by staining with the fluorescent DNA-binding compound, Hoechst 33258 (Esteban, 1977). While factories were observed to form after HU reversal in the cytoplasm of infected cells treated with cycloheximide, they were sparse and failed to increase in size with time, as was observed to occur in cells continuously synthesizing protein (Fig. 6). While this is only a qualitative measure, the method did provide evidence that viral DNA synthesis was inhibited as shown by the failure of DNA to

HOLOWCZAK

accumulate within the factories when protein synthesis was interrupted. We concluded that incorporation of [3H]thymidine into acid-insoluble material, even after treatment of infected cells with cycloheximide, does provide meaningful information concerning viral DNA replication. This is particularly true after reversal of a HU block since viral DNA synthesis can be studied after host DNA synthesis is significantly inhibited (Pogo and Dales, 1971). Dahl and Kates (1970) suggested that the release of viral DNA from virosomes requires the synthesis of proteins involved in the initial stage of virion assembly. We have recently demonstrated that viral DNA can be released from complexes by digestion in vitro with S-l nuclease but not pronase or RNase (Esteban and Holowczak, 1977b). These results indicated that ssDNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes. A variety of DNase activities capable of hydrolyzing native DNA at an alkaline pH and denatured DNA at a neutral and low pH have been detected in cells infected with vaccinia virus (reviewed in Moss, 1974). One can therefore speculate that the protein required for release of newly replicated vaccinia genomes from complexes in uivo may be a nuclease; the acid DNase, a late viral enzyme (McAuslan and Kates, 1967), could perform such a function. Using rifampin, Esteban (1977) was able to show that mature, crosslinked vaccinia DNA molecules were synthesized even when viral assembly was prevented. We have demonstrated here that release of viral genomes from complexes is not inhibited by rifampin. We conclude therefore that the replication of viral genomes, including their release from complexes, occurs independently of viral assembly. ACKNOWLEDGMENTS The expert technical assistance of Ms. Domenica Bucolo is gratefully acknowledged. This investigation was supported hy Public Health Service Research Grant CA-11027-08 from the National Cancer Institute. J. A. Holowczak is a recipient of Public Health Service Research Career Development Award CA-70458 from the National Cancer Institute.

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