INTRODUCTION

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Herpes simplex virus (HSV) replicates in both cycling and noncycling cells, including terminally differentiated neurons. HSV replication, however, requires cellular functions associated with cell cycle progression. Thus, HSV replicates more efficiently in actively dividing than growth-arrested cells. This difference in replication efficiency is especially prominent among viral mutants which lack specific viral functions such as those provided by the regulatory proteins ICP0 and VP16 or by enzymes involved in nucleotide metabolism, such as thymidine kinase (TK) or ribonucleotide reductase (9, 16, 25). The dependence on cell cycle-activated cellular functions is further evidenced by the fact that HSV cannot replicate in temperature-sensitive (ts) cells growth-arrested in G₀/G₁ at the nonpermissive temperature (78, 86). At the molecular level, several cellular proteins, DNA binding activities, and other cellular activities normally involved in cell cycle progression, including HCF, E2F, cdk-2, cyclin D3, and DNA polymerase (Pol) , are (i) required for viral gene expression, (ii) activated during HSV infection, (iii) localize to the sites of viral replication, and/or (iv) interact physically with HSV proteins (26, 33, 37, 45, 80, 83, 84). Furthermore, we have recently shown that cellular cyclin-dependent kinases (cdks) are required, directly or indirectly, for the transcription of HSV immediate-early (IE) and early (E) genes and hence for viral replication (44, 70, 71).

Cellular cdks comprise a family of serine-threonine protein kinases which, at present, includes nine members. The activities of these enzymes are regulated by association with specific cyclin partners, phosphorylation, and interaction with cdk inhibitors (cdis) such as p21^CIP, p27^KIP1, p16^INK4a, and p15^INK4b, whose expression is itself tightly regulated. Thus, the most active form of a typical cdk occurs when it is associated with its appropriate cyclin partner, phosphorylated by the cdk-activating kinase at specific residues, dephosphorylated by Cdc25 at other specific residues, and not complexed with cdis (reviewed in references ;[31, 39, 54, and 65]). Of all known cdks, cdk-1, cdk-2, cdk-3, cdk-4, cdk-6, and cdk-7 are recognized as key cell cycle regulators in that they directly and indirectly regulate DNA synthesis, mitosis, and transcription of genes whose products are required for cell cycle progression. Moreover, inhibition of the activity of cdk-1, cdk-2, or cdk-3 results in cell cycle arrest (79). With regard to the involvement of cdks in transcription, cdk-7 and cdk-8 phosphorylate directly the components of the cellular transcriptional complex whereas cdk-1, cdk-2, cdk-4, and cdk-6 directly and indirectly regulate the activity of several transcription factors (4, 13, 19, 20, 24, 34, 47, 49, 52, 66, 67, 73, 74, 76, 79, 85). With regard to the involvement of cdks in DNA synthesis, cdk-2 is required for this process in that DNA synthesis does not occur if cdk-2 or one of its cyclin partners, cyclin A, is blocked with specific antibodies (10, 22, 62, 68, 88). Mechanistically, activation of cdk-2 is required for expression of many cellular DNA replication proteins and cdk-2 phosphorylates and activates several of the cellular proteins that are required for DNA synthesis (6, 10, 11, 40, 48).

In addition to their roles in cellular functions, cdks are known to be involved directly or indirectly in viral DNA synthesis. For example, simian virus 40 and polyomavirus large T antigens, which initiate viral DNA replication, are activated by cdk-2-mediated phosphorylation (12, 28, 51). cdks are also required for human cytomegalovirus HCMV DNA replication in that cdk inhibitors block HCMV DNA synthesis but do not inhibit the expression of IE or E proteins (7).

Our previous studies have shown that, like other DNA viruses, cdks are also required for HSV replication (44, 70, 71). Specifically, we have shown that HSV replication is blocked by two cdk inhibitors, roscovitine (Rosco) (17, 56) and olomoucine (Olo) (1, 8, 23, 72, 81). Rosco and Olo specifically inhibit a subset of cdks which includes cdk-1, cdk-2, and cdk-5 but not cdk-4 or cdk-6. In vivo, Rosco inhibits cell cycle progression at concentrations between 10 and 100 µM, depending on the cell line used (2, 38, 56). This inhibition of cell cycle progression has been determined to be mediated by inhibition of cdk-1 and cdk-2 activities (2, 56). Other inhibitors which are specific for protein kinases or cell cycle progression but which do not inhibit cdks had no significant effect on HSV replication (70). With respect to individual viral functions that require cdks, Rosco inhibited the transcription of IE and E genes as well as viral DNA synthesis (44, 70, 71). As noted in these previous studies, however, inhibition of viral DNA synthesis could have resulted exclusively from the block in expression of IE and E proteins. Alternatively, HSV DNA synthesis could have been inhibited as a result of inhibition of IE and E protein expression and inhibition of cdks required directly for viral DNA synthesis. Although we were unable to discriminate between these two alternatives in the previous studies, we noted that Rosco inhibited HSV replication even when added to infected cells at 6 h postinfection (p.i.), when E proteins should already have been synthesized (71). These unexpected results prompted us to ask whether Rosco also inhibits essential viral replication functions, such as DNA synthesis, that occur after E proteins have been expressed.

In the experiments described herein, we examined the effects of Rosco on HSV DNA synthesis under conditions in which IE and E proteins had already been synthesized. We found that Rosco inhibits HSV replication after release from a block in viral DNA synthesis induced by phosphonoacetic acid (PAA) or following a shift-down of cells infected with HSV-1 DNA ts mutants from the nonpermissive to the permissive temperature. Under these conditions, Rosco-induced inhibition of viral replication was shown to occur at the level of viral DNA synthesis whereas expression of E proteins was not affected. Based on these observations, we conclude that HSV DNA synthesis, as well as transcription of IE and E genes, requires the activities of Rosco-sensitive cellular cdks.

	MATERIALS AND METHODS

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Cells, virus, plasmids, and drugs. Methods used for the growth and maintenance of Vero cells and for the propagation of a low-passage (p9), plaque-purified, stock of HSV-1, strain KOS, have been described previously (70). DNA ts mutants of HSV-1 KOS, tsA1, tsA15, tsD9, and tsP23 (41, 63, 69), and plasmids prpTK and prp8 have also been described (43).

Infections. Vero cells (10⁵) were infected at the indicated multiplicities with HSV-1 KOS as previously described (70, 71). Where indicated, medium overlying infected cells was replaced with fresh drug-containing or control (drug-free) medium. Viral titers at specific times p.i. were determined by standard plaque assays (70, 71).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Effect of Rosco on HSV replication when added at 3-h intervals after infection. Vero cells were infected with 2.5 PFU of HSV-1 per cell. After 1 h of adsorption, the cells were washed and overlaid with medium containing no drug (control) or 100 µM Rosco. At 3, 6, 9, 12, 15, 18, and 21 h p.i., medium was removed and replaced with medium containing 100 µM Rosco (dotted lines, solid triangles). Changes from drug-free to Rosco-containing medium are indicated by the arrows. A second set of infected monolayers was left in drug-free medium (solid line, solid squares). At 1, 3, 6, 9, 12, 18, 21, and 24 h p.i., cultures were harvested and viral titers were determined by a standard plaque assay. Viral titers are plotted as a function of time p.i. Each time point indicates the average of two independent experiments.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   The inhibition of HSV replication by Rosco added at the time of release from a 12-h PAA block is multiplicity dependent. (A) Vero cells were infected with HSV-1 at the indicated multiplicities of infection, washed, and overlaid with medium containing 100 µg of PAA per ml or 100 µM Rosco. Infected monolayers were harvested at 24 h p.i., and viral replication was calculated by dividing the viral titer at 24 h p.i. by the adsorbed PFU. (B) Vero cells were infected with HSV-1 at the indicated multiplicities, washed, and overlaid with medium containing 100 µg of PAA per ml. At 12 h p.i., medium was removed from infected monolayers and replaced with fresh medium containing no drug (PAA/C), 100 µg of PAA per ml (PAA/PAA), or 100 µM Rosco (PAA/RO). At 24 h after the change of medium, cells were harvested and viral titers were determined by a standard plaque assay. Viral yields at 24 h p.i. are plotted against the multiplicities of infection (moi). For reference, the dotted line indicates the number of PFU/106 cells in inocula at each multiplicity. The average and range of two independent experiments are shown for each time point.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   The inhibition of HSV DNA synthesis by Rosco added at the time of release from a 12-h PAA block is multiplicity dependent. (A) Vero cells were infected at the indicated multiplicities (MOI) with HSV, washed, and overlaid with medium containing 100 µg of PAA per ml. At 12 h p.i. medium was removed and fresh medium containing no drug (PAA 12  C 24), 100 µg of PAA per ml (PAA 12  PAA 24), or 100 µM Rosco (PAA 12  Ro 24) was added. At 24 h after the change of medium, cells were harvested and the amounts of viral DNA were determined by slot blot analysis. (B) After quantitation of the blots presented in panel A using a Molecular Dynamics PhosphorImager system, the fold increase in viral DNA replication was calculated by dividing the amount of viral DNA detected at 12 h postrelease by the amount detected immediately after infection and subtracting 1, such that a total block in viral DNA replication by the secondary drug would be indicated by 0-fold increase. The fold increase in DNA replication was then plotted against the multiplicity (MOI). The results from one of two repeat experiments are presented.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Effect of Rosco and PAA on the steady-state levels of selected viral transcripts when drug is added at the time of release from a 12-h PAA block. Vero cells were infected with 2 PFU of HSV per cell, and medium was replaced as described in the legend to Fig. 3B. Immediately before release (0) and at 4, 8, and 16 h after release (hpr) from the PAA block and addition of the secondary drug, cells were harvested and total RNA was extracted. RNA extracted from mock-infected cells served as a negative control (MI). Levels of ICP0 (IE), ICP8 (E), and TK (E) transcripts were determined by RNase protection assays.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Inhibition of HSV DNA synthesis by Rosco in the absence or presence of CHX added at the time of release from a 12-h PAA block. (A) Vero cells were infected with 2 PFU of HSV per cell, incubated in the presence of 100 µg of PAA per ml for 12 h, and released from the block as described in the legend to Fig. 3A, except that CHX alone or together with the secondary drug was added to one set of infected monolayers at the time of release (+ CHX). At 1 h p.i. (HPI) (11 h postrelease [HPR]) immediately before release (0 h postrelease; 12 h p.i.), and at 12, 16, and 20 h postrelease (24, 28, and 32 h p.i., respectively), cells were harvested and total DNA was extracted. Levels of viral DNA were determined by slot blot hybridization at the indicated times. (B and C) Viral DNA in the blots in panel A were quantitated using a Molecular Dynamics PhosphorImager system, and the fold increase in viral DNA synthesis after release from the PAA block in the absence (B) (CHX) or presence (C) (+CHX) of CHX was calculated by dividing the amounts of viral DNA at the indicated times postrelease by the amounts of viral DNA before release and subtracting 1, such that total inhibition of DNA replication by the secondary drug would be represented by 0-fold increase. The fold increase in DNA synthesis is plotted against hours postrelease (hpr). Data from one of two repeat experiments is presented.

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Replication of four HSV ts mutants after the shift-down from the nonpermissive to the permissive temperature in the presence of Rosco. Vero cells were infected at the nonpermissive temperature with 2.5 PFU of the indicated HSV ts mutants per cell. At 6 h p.i., infected cultures were transferred to the permissive temperature in the absence of drug (Control) or in the presence of 100 µM Rosco. One culture infected with each ts mutant was harvested immediately before the shift-down. At 24 h after the shift-down, the remaining infected monolayers were harvested and viral replication was monitored by a standard plaque assay. Fold viral replication after release was determined by dividing the titers at 24 h postrelease by the titers measured before release and subtracting 1.

View larger version (1439K): [in this window] [in a new window]   FIG. 7.   HSV DNA synthesis after the shift-down from the nonpermissive to the permissive temperature in the presence of Rosco added at the time of the shift-down. (A) Vero cells were infected with HSV tsA15 at the nonpermissive temperature and shifted down in the absence of drug or in the presence of 100 µM Rosco or 100 or 400 µg of PAA per ml (Control, RO, PAA100, and PAA400, respectively), as described in the text. At 1 h p.i. (HPI) (5 h postrelease [HPR]), immediately before release (6 h pi/0 hpr) and at 15 and 24 h after release (21 or 30 h p.i., respectively), cells were harvested and DNA was extracted. DNA was also extracted from mock-infected cells for comparison (MOCK). Levels of viral DNA synthesis were determined by slot blot analysis. For comparison, a set of infected monolayers was incubated at the permissive temperature throughout the experiment. (B) The slot blots shown in panel A were quantitated using the ImageQuant software package (Molecular Dynamics), and the fold increase in DNA synthesis was calculated by dividing the amount of viral DNA at a given time point by the amount of viral DNA detected immediately before release and subtracting 1, such that complete inhibition of viral DNA replication after release would be indicated by 0-fold increase. The fold increase in DNA replication is plotted against hours postrelease (hpr). Data from one of two repeat experiments are presented.

View larger version (57K): [in this window] [in a new window]   FIG. 8.   Expression of E gene products by tsA15 in the presence of Rosco added at the time of the shift-down. (A) Vero cells were infected with tsA15 at the nonpermissive temperature and shifted down to the permissive temperature as described in the legend to Fig. 7. Immediately before release (Pre) or 16 h after the shift-down in the absence of drug or in the presence of 100 µM Rosco or 100 or 400 µg of PAA per ml (C, RO, P1, and P4, respectively), cells were harvested and RNA was extracted. Steady-state levels of the transcripts of the genes encoding ICP8 or TK were evaluated by RNase protection assays. (B) Vero cells were infected with tsA15 at the nonpermissive temperature and shifted down to the permissive temperature as described for panel A, except that methionine-free medium supplemented with 50 µCi of [35S]methionine per ml and the indicated drugs (C, RO, P1, or P4) were added at the time of the shift-down. Infected cells were harvested at 16 h p.i., and viral proteins were resolved in a sodium dodecyl sulfate-polyacrylamide gel. Molecular weights (in thousands) are indicated on the right. The ICP nomenclature was used, such that VP16 is designated ICP25/26. HSV late proteins (1 and 2) are indicated on the left of the gel by open arrowheads. Viral IE and E proteins are indicated on the right of the gel by solid arrowheads. (C) Infected cells were infected at the nonpermissive temperature, shifted down, and labeled with [5S]methionine as described in the legend to panel B, except that the cells were harvested at 24 h after the shift-down. Molecular weights, ICP nomenclature, and HSV IE, E, and L proteins (1 and 2) are labeled as in panel B.

Probes, RNase protection assays, and viral DNA synthesis assays. The synthesis of riboprobes, RNase protection assays, and quantitation of viral DNA by slot blot analysis were performed as previously described (70, 71).

Metabolic labeling. For metabolic labeling experiments (see Fig. 8), cells were infected with 5 PFU of tsA15 per cell at the nonpermissive temperature and shifted down to the permissive temperature in methionine-free medium containing no drug, PAA (100 or 400 µg/ml, as indicated) or Rosco (100 µM), and 50 µCi of [³⁵S]methionine per ml.

	RESULTS

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Rosco inhibits HSV replication when added to infected cells at 3, 6, 9, or 12 h p.i. As a first step in determining whether Rosco-sensitive cdks are required for essential viral functions that occur after E protein synthesis, we asked whether addition of Rosco at different times after infection inhibited HSV replication. If Rosco inhibits only expression of IE and E gene products, addition of the drug after 6 h p.i., when IE and E gene products have already been synthesized, should have little effect on viral replication. In contrast, if the drug inhibits an essential viral function that occurs after synthesis of E gene products, Rosco should inhibit HSV replication even when the drug is added after 6 h p.i. To address this question, Vero cells were infected with 2.5 PFU of HSV-1 per cell and one infected monolayer was harvested every 3 h while medium from another infected monolayer was replaced with medium containing 100 µM Rosco. We selected this concentration because we have previously determined that 100 µM Rosco is required to block cell cycle progression completely and HSV replication in Vero cells (70). All monolayers in Rosco-containing medium and the single monolayer remaining in control medium were harvested at 24 h p.i. Levels of total infectious virus were determined by plaque assays.

Rosco inhibits HSV replication after release from a 12-h PAA block. Based on previously published observations (71) and on the findings shown in Fig. 1, we hypothesized that cellular cdks are required for essential viral functions that occur after E gene expression. Because viral DNA synthesis occurs immediately after, or concomitant with, E gene expression, we hypothesized that viral DNA synthesis may be an essential viral function that requires cdks. As a first test of this hypothesis, we asked whether Rosco can inhibit HSV replication after release from a PAA block. PAA is a well-characterized inhibitor of HSV DNA polymerase (3, 29, 36, 42, 53, 61). In the presence of PAA, viral IE and E proteins are synthesized but the activity of the viral DNA Pol (an essential E protein) is directly inhibited by the drug. Consequently, E proteins are expressed but viral DNA is not synthesized.

Inhibition of HSV DNA synthesis by Rosco added at the time of release from a 12-h PAA block is multiplicity dependent. We next determined the level of HSV DNA synthesis that occurs after release from a 12-h PAA block into Rosco-containing medium. As controls, we also measured the efficiency of DNA synthesis after release from a 12-h PAA block in drug-free medium (control) or in the presence of PAA. For these experiments, we used the same range of multiplicities used in the experiments in Fig. 2. Vero cells were thus infected with 0.1 to 10 PFU of HSV-1 per cell, and infected cells were treated with 100 µg of PAA per ml for 12 h as described above. Infected cells were then released from the PAA block in the absence of drug or in the presence of PAA or Rosco. Immediately before or 24 h after the change of medium, cells were harvested, DNA was extracted, and viral DNA was quantitated as previously described (70, 71).

Levels of E transcripts are unaffected when Rosco is added after release from a 12-h PAA block. The inhibition of HSV DNA synthesis induced by Rosco after release from a 12-h PAA block might have been secondary to a decrease in the levels of the viral E proteins required for viral DNA synthesis. Because metabolically labeled viral proteins were not detected in amounts that allowed reliable quantitation at low multiplicities (<5 PFU/cell) (reference ;[59] and data not shown), we evaluated levels of viral transcripts as an indirect measure of levels of viral proteins. Notably, we have shown previously that Rosco does not inhibit the translation of viral proteins after their mRNAs have been synthesized (71). Vero cells were infected with 2 PFU of HSV-1 per cell, and infected cells were incubated in the presence of 100 µg of PAA per ml and released from the PAA block into no drug, PAA, or Rosco (Fig. 4A), as described above. Immediately before or 4, 8, or 16 h after the change of medium, cells were harvested and RNA was extracted and quantitated by RNase protection assays. A multiplicity of 2 PFU/cell was selected because it was the highest multiplicity at which Rosco inhibits viral DNA synthesis efficiently after release from a 12-h PAA block (Fig. 3).

Rosco inhibits HSV DNA synthesis when added after release from a 12-h PAA block, even in the absence of new protein synthesis. Because a reversible PAA block can be achieved only at relatively low multiplicities (36, 61) and because most of the essential viral DNA synthesis proteins are expressed at very low levels (e.g., UL5, UL8, UL9-OBP-, and UL52 [59]), it was not possible to measure accurately the levels of all viral DNA replication proteins synthesized in cells infected and released from a PAA block. As an alternative, we evaluated HSV DNA synthesis after release from a PAA block in the presence of 50 µg of CHX per ml and either 100 µg of PAA per ml or 100 µM Rosco. Addition of CHX at the time of release from a PAA block results in inhibition of new viral protein synthesis. Therefore, in these experiments, viral DNA is synthesized after release from the PAA block by viral proteins synthesized before release. Hence, Rosco could have no effect on viral gene expression, which would have occurred before addition of the drug. If the inhibition of viral DNA synthesis produced by Rosco in the PAA release experiments was exclusively a consequence of inhibition of E gene expression, Rosco should not inhibit viral DNA synthesis beyond the inhibition resulting from the addition of CHX at the time of release. In contrast, if inhibition of viral DNA synthesis was at least partially due to a requirement for enzymes that are sensitive to inhibition by Rosco, CHX and Rosco together should inhibit viral DNA synthesis to a greater extent that should CHX alone. We therefore performed experiments in which no drug, Rosco, CHX alone, Rosco and CHX, or PAA and CHX were added to infected cells at the time of release from the primary 12-h PAA block.

Replication of DNA HSV ts mutants is inhibited by Rosco added at the time of shift-down from the nonpermissive to the permissive temperature at 6 h p.i. To obtain independent evidence to support or refute the hypothesis that Rosco inhibits viral DNA replication in the presence of E proteins, we asked whether Rosco added at the time of shift-down from the nonpermissive to the permissive temperature inhibits replication of HSV mutants with ts mutations in genes encoding E proteins required for viral DNA synthesis. For this purpose, we tested four mutants, tsA1, tsA15, tsD9, and tsP23. The mutations in tsA1 and tsA15 have been mapped to the ICP8 gene, and the mutation in tsD9 has been mapped to the catalytic subunit of the viral DNA polymerase (63, 69). The mutation in tsP23 has not been mapped definitely, but available evidence indicates that it, too, maps to the viral DNA polymerase gene (42).

Rosco inhibits DNA synthesis by tsA15 following the temperature shift-down. We next asked whether the inhibition of viral replication observed in the experiments described above was a result of inhibition of viral DNA synthesis. In these experiments, we chose to study mutant tsA15 because (i) the mutation in this virus has been mapped, (ii) reversal of the block in viral replication is efficient following the shift-down, and (iii) Rosco inhibited its replication efficiently after the shift-down (Fig. 6). We used a multiplicity of 5 PFU/cell because expression of E gene products and of viral DNA synthesis were to be examined in parallel. We had determined in preliminary experiments that 5 PFU/cell is the lowest multiplicity that permits reliable quantitation of E protein synthesis by metabolic labeling in our hands (data not shown).

tsA15 gene expression when shifted down to 34°C in the presence of Rosco after a 6-h incubation at 39.5°C. If inhibition of viral DNA synthesis by Rosco after the shift-down was exclusively due to inhibition of E gene expression, levels of E transcripts and proteins should be significantly reduced under the same conditions in which viral DNA synthesis is inhibited. If inhibition of viral DNA synthesis was mediated at least partially by a posttranslational mechanism, levels of E transcripts and proteins might not be significantly inhibited under conditions in which viral DNA synthesis was. To test these hypotheses, we evaluated the levels of two E transcripts and a number of E proteins following a shift-down of tsA15-infected cells from 39.5 to 34°C. Specifically, we choose to examine transcripts of the gene encoding ICP8, because it is the gene mutated in tsA15, and the gene encoding TK, because it is an E protein involved in an aspect of viral DNA replication distinct from the function of ICP8 (55).

	DISCUSSION

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Collectively, the results of these studies demonstrate that in addition to transcription of IE and E genes, HSV DNA synthesis requires cellular cdks or perhaps some other cellular protein(s) not yet known to be sensitive Rosco. Four points are critical to the interpretation of the results described herein. First, a structural isomer of Rosco, which does not inhibit cdks, does not inhibit HSV replication (70). Thus, the inhibition of viral DNA synthesis by Rosco observed in the experiments reported herein is not mediated by the core structure of the compound but, rather, by its kinase-inhibitory activity. Second, the concentration of PAA used in the PAA reversal experiments, 100 µg/ml, is acknowledged to be below the concentration required to achieve a complete block in HSV DNA synthesis (200 to 400 µg/ml) (3, 36, 61); (Fig. 3, 5, and 7). However, concentrations of PAA that completely inhibit HSV DNA synthesis will block viral replication irreversibly (36, 61]; L. Schang and P. Schaffer, unpublished data), and hence could not be used in the PAA reversal experiments. Since Rosco inhibited HSV DNA synthesis when added at the time of release from a partial PAA block (Fig. 3 and 5), we are confident that the inhibition observed in these experiments is an underestimate of the actual inhibitory effect of Rosco on viral DNA synthesis. Third, the three known or putative viral kinases are all nonessential in vitro (64), and we have yet to detected any HSV Rosco-resistant mutant (;[70]; Schang and Schaffer, unpublished). Thus, the inhibitory effects of Rosco observed in the experiments described in this paper are not likely to be the result of inhibition of one or more of the three virally encoded kinases. Finally, it must be emphasized that the design of the experiments presented in this paper required rapid inhibition of cellular cdk activities at specific times p.i. Only chemical cdk inhibitors act with the required speed, and consequently only chemical (as opposed to protein) cdk inhibitors could be used in the experiments presented in this paper.

Effects of Rosco on the levels of HSV-1 E gene transcription. We had observed previously that Rosco inhibits accumulation of transcripts of viral E genes after release from a 6-h CHX block but not when added at 6 h p.i. in the absence of a previous block (71). Moreover, steady-state levels of IE transcripts were lower in the presence of Rosco (70, 71). In the experiments presented herein, the levels of two E transcripts remained nearly constant when Rosco was added after release from a PAA block (Fig. 4) or after a shift-down from the nonpermissive to the permissive temperature (Fig. 8). We observed, however, that the levels of ICP8 transcripts were somewhat lower in the presence than in the absence of Rosco. The nearly constant levels of E transcripts following the addition of Rosco may be due to the long intrinsic half-lives of E transcripts (32), especially since E transcripts are stabilized even further in the absence of vhs (60), a late gene not expressed in the absence of DNA synthesis. Notably, in the experiments in which Rosco inhibited accumulation of E transcripts (Fig. 7 of reference ;[71]), Rosco was added before initiation of E gene transcription. In contrast, in the experiments in which the levels of E transcripts remained constant in the presence of Rosco (Fig. 4 and 8; Fig. 3 of reference ;[71]), Rosco was added several hours after transcription of E genes had begun. It is thus possible that only initiation of E gene transcription is sensitive to inhibition by Rosco. In this scenario, the constant levels of E transcripts observed in the experiments in Fig. 4 and 8 would be the result of continuation of transcription initiated before addition of the drug. Experiments are in progress to determine whether the constant levels of E transcripts in the presence of Rosco added at 6 h p.i. (71) or at the time of release from blocks in viral DNA synthesis are due to (i) the extended half-lives of these transcripts or (ii) continuous transcription in the presence of the drug. The reduced steady-state levels of ICP0 transcripts detected in the presence of Rosco could be explained by destabilization of these transcripts or by inhibition of transcription in the presence of the drug. Because the levels of ICP0 mRNA decreased in the presence of Rosco with a half-life consistent with the half-life of ICP0 transcripts when transcription is inhibited with amantadine (32, 60), we conclude that inhibition of transcription rather than destabilization is the most likely explanation for the reduced levels of ICP0 mRNA in the presence of Rosco.

Effects of Rosco on HSV DNA synthesis. Based on a comparison of the findings presented in Fig. 5, and 7, Rosco appeared to inhibit DNA synthesis more efficiently in ts mutant-infected cells after the shift-down to the permissive temperature than in wild-type-virus-infected cells after reversal of a PAA block (compare Fig. 5 and 7). Several differences between the two experimental systems could explain these apparent differences in efficiency. First, the block in DNA synthesis (for tsA15) at 39.5°C is clearly tighter than the block in DNA synthesis achieved by 100 µg of PAA per ml for wild-type KOS. Consequently, if Rosco inhibited DNA synthesis to the same extent after release from the blocks induced by PAA or by incubation at 39.5°C (for instance, if the efficiency of viral DNA synthesis after release were only onefold above the efficiency of viral DNA synthesis before release), the observed inhibition would be greater in the shift-down than in the PAA release experiments. Moreover, reversal of the block in DNA synthesis after the shift-down is significantly more efficient than the reversal which followed removal of PAA (compare Control in Fig. 5 and 7). Consequently, if viral DNA replicated to the same extent in the presence of Rosco added after the shift-down from the nonpermissive temperature as after release from the PAA block, the inhibition of DNA synthesis measured as a fraction of DNA replication after release in the absence of secondary drug would be significantly different in the two experimental models.

Potential roles of cdks in HSV DNA synthesis. The observed requirement for cdks in HSV DNA synthesis can be explained by three potential mechanisms of action: (i) cdks may be required to activate viral DNA replication proteins, (ii) HSV DNA synthesis may require cellular DNA replication proteins (several of which are known to be activated by cdks), or (iii) HSV DNA synthesis may be mechanistically linked to viral transcription (which is also known to require cdks).

cdks that may be required for viral DNA synthesis. From the results of the experiments presented in this and previous papers (70, 71), we conclude that transcription of HSV IE and E genes and viral DNA replication all require Rosco-sensitive enzymes. Given that Rosco inhibits cdk-1, cdk-2, cdk-5 (56), and cdk-7 (L. Schang, R. Shiekhattor, and P. Schaffer, unpublished observations) and probably also cdk-3 (70), different cdks may be required for different viral functions. Moreover, other as yet unknown cellular targets of Rosco may exist. Therefore, we have yet to determine which one(s) of the Rosco-sensitive cdks, or other kinases, is specifically required for each of the Rosco-sensitive HSV replication functions. With respect to viral DNA replication, however, we hypothesize that cdk-2 may be the cellular kinase that is (i) required for this viral function and (ii) sensitive to inhibition by Rosco. This hypothesis is based on the fact that cdk-2 is considered a central player in cellular DNA replication. Thus, cdk-2 localizes to cellular DNA replication forks (10) and its activity is required to initiate replication of cellular DNA (48). At the molecular level, cdk-2 phosphorylates and activates a number of DNA replication proteins, including cellular ribonucleotide reductase, RF-A, DNA Pol , Pol , proliferating-cell nuclear antigen, and/or topoisomerase I and II (18, 82). Moreover, cdk-2-mediated phosphorylation of proteins that regulate DNA replication, such as HsCdc6, is also required for cellular DNA replication (40). Finally, the expression of many enzymes required for cellular DNA replication (including DNA ligase 1 and DNA Pol ) is dependent on cdk-2 activity (18, 52), and, as noted above, some of these cellular proteins may also be required for HSV DNA synthesis (27, 46, 50, 75).

HSV replication functions that occur after DNA synthesis are likely to be sensitive to inhibition by cdk inhibitors. The fact that Rosco also inhibits HSV replication when added at times after the time when the majority of viral DNA should already have been synthesized (i.e., 12 h p.i.) has not escaped our notice. Considering that three viral processes (transcription of IE genes, transcription of E genes, and viral DNA synthesis) require enzymes sensitive to inhibition by Rosco, it seems likely that viral functions that occur after DNA synthesis may also require Rosco-sensitive enzymes. Interestingly, a structural L protein of another alphaherpesvirus, varicella-zoster virus, was recently shown to be phosphorylated by cellular cdk-1, which is sensitive to Rosco inhibition (87). Although cdk-mediated phosphorylation of L proteins may well occur during HSV infection, this hypothesis remains to be tested.