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and Deborah H. Spector*
Molecular Biology Section and Center for Molecular Genetics, University of California, San Diego, La Jolla, California
Received 18 February 2004/ Accepted 3 June 2004
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A second locus of IE transcription is UL36-38. This region includes three promoters and gives rise to at least five transcripts (Fig. 1C). One of the IE promoters directs the synthesis of several spliced 3.2- to 3.4-kb RNAs (UL37 and UL37M) that are present in small amounts only at IE times. It also is the promoter for an abundant 1.7-kb unspliced RNA that encodes the UL37 exon 1 (UL37X1) gene product, which is present throughout the infection. The cleavage-polyadenylation site for the UL37X1 RNA is located 8 nucleotides upstream of the splice acceptor site for exon 2 of the UL37 and UL37M RNAs. The second IE promoter is responsible for the synthesis of a 1.65-kb spliced RNA (UL36) that increases in abundance at early times and is 3' coterminal with the UL37 spliced RNAs (36, 66, 67). In addition, another promoter within UL37X1 directs the synthesis of an abundant early transcript of 1.35 kb (UL38) that is 3' coterminal with the UL37X1 RNA. The start site of the UL38 transcript is 70 nucleotides downstream of the splice donor site for the UL37 and UL37M RNAs (66, 67).
The UL37X1, UL37, and UL36 proteins have regulatory activity, and the locus encoding these proteins is required for HCMV DNA replication in a transient complementation assay (17, 30, 50, 51, 62, 74). In addition, the UL37X1 protein and the UL37 and UL37M glycoproteins have a common amino terminus of 162 amino acids. Within this common domain are a hydrophobic signal sequence (amino acids 1 to 22) and an acidic domain (amino acids 81 to 108). The UL37 glycoprotein also has 17 N-glycosylation sites, a basic domain, a transmembrane region, and a cytosolic tail. UL37X1 encodes an antiapoptotic protein (viral mitochondrion-localized inhibitor of apoptosis) that prevents release of cytochrome c from the mitochondria (16, 28, 41).
In addition to the viral proteins, many cellular proteins play an important role in the replication of HCMV. Some of these proteins also play pivotal roles in the regulation of the cell cycle. The cell cycle includes four phases (G1, S, G2, and M), and both entry into and exit from these phases are largely controlled by regulating the activity of the cyclin-dependent kinases (cdks) and expression of their cyclin partners (for review, see references 23 and 54). Multiple studies have addressed the effect of HCMV infection on the expression of these cyclins and the progression of the cell cycle (6-12, 14, 15, 21, 25, 26, 31, 33, 34, 38, 40, 42, 45-47, 49, 59, 69-71). The general picture that emerges from these studies is that HCMV activates or induces the expression of many host cell proteins to create a cellular environment that is optimal for gene expression and DNA replication; however, the virus inhibits selective host cell functions to ensure that its own replication is favored over that of the host. The net effect is that the cell cycle is blocked prior to the initiation of cellular DNA synthesis.
In this study, we used the drugs Roscovitine and Flavopiridol to assess the role of cyclin-dependent kinases in HCMV infection. Roscovitine is a purine-derived compound that specifically inhibits the activity of cdk1/cyclin B, cdk2/cyclin E, cdk2/cyclin A, cdk5/p25 (expressed in neural cells), cdk7/cyclin H, and cdk9/cyclin T1 (P-TEFb) with a 50% inhibitory concentration (IC50) of approximately 0.7 µM in in vitro kinase assays; cdk4/cyclin D and cdk6/cyclin D are not inhibited (20, 22, 43, 61, 68). Flavopiridol, a flavanoid, is another specific inhibitor of cyclin-dependent kinases that acts as a competitor with ATP to inhibit cdk1/cyclin B (IC50 = 30 to 40 nM in in vitro kinase assays), cdk2/cyclin A and cdk2/cyclin E (IC50 = 100 nM), cdk4/cyclin D (IC50 = 20 to 40 nM), cdk6/cyclin D (IC50 = 60 nM), and cdk7/cyclin H (IC50 = 110 to 300 nM). It is also a potent inhibitor of P-TEFb that binds to P-TEFb with 1:1 stoichiometry and is not competitive with ATP (for review, see reference 18).
Both of these drugs are being used in clinical trials as potential anticancer agents and appear to have low toxicity. The inhibition of the replication of the herpesviruses herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, varicella-zoster virus, and HCMV as well as human immunodeficiency virus type 1 by Roscovitine has led to the proposal that productive infection by these viruses requires the activity of one or more cyclin-dependent kinases (for review, see reference 60). Flavopiridol also inhibits human immunodeficiency virus type 1 replication primarily through its effect on P-TEFb (IC50 < 10 nM).
In a prior study on HCMV, Roscovitine was found to inhibit viral DNA synthesis in density-arrested human lung fibroblasts and in a transformed astrocytoma/glioblastoma cell line (U373) (9). The authors concluded from the results of transient expression assays with a dominant negative cdk2 expression vector in infected U373 cells that this cyclin-dependent kinase was required for late gene expression; however, the low percentage of cells transfected with the vector (<5%) coupled with the inability to infect more than 30% of the cells complicated the interpretation of these experiments.
In this work, we sought to determine whether the requirement for cyclin-dependent kinase activity was restricted to the early period of infection and to decipher what molecular processes were affected by the absence of these kinases. We have shown that the addition of Roscovitine at the beginning of the infection resulted in altered processing and accumulation of UL122-123 and UL37 transcripts and inhibition of the expression of selected viral early genes. These effects did not require de novo protein synthesis or degradation of proteins via the proteasome. Delaying the addition of Roscovitine until 6 h p.i. abolished these effects and viral replication proceeded through late gene expression, but viral titers were still significantly reduced. We also demonstrated that inhibiting cyclin-dependent kinase activity with the drug Flavopiridol had similar effects on IE and early gene expression.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell synchronization and infections. Cells were synchronized in the G0 phase by allowing them to grow to confluence as previously described (59). Three days after confluence, the cells were trypsinized, replated at a lower density to allow progression into the cell cycle, and infected at a multiplicity of infection (MOI) of 3 to 5 with HCMV Towne or mock infected with tissue culture supernatants. At designated times p.i., Roscovitine (Calbiochem) or Flavopiridol (gift from J. Brady, National Institutes of Health) was added to the medium. The Roscovitine stock solution was 10 mM in dimethyl sulfoxide, and the Flavopiridol stock solution was 0.1 mM in dimethyl sulfoxide. Control samples were treated with appropriate volumes of dimethyl sulfoxide. At various times p.i., cells were washed with phosphate-buffered saline, scraped or trypsinized, and processed as described.
Cytotoxicity assays. The toxic effects of Roscovitine on HFF cells were evaluated with the LIVE/DEAD viability/cytotoxicity assay kit (Molecular Probes) according to the manufacturer's protocol. This assay uses two-color fluorescence to simultaneously determine the presence of live and dead cells in a culture. In live cells, the nonfluorescent cell-permeable calcein acetoxymethyl (AM) dye is converted by intracellular esterase activity to calcein, which fluoresces green. Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of red fluorescence upon binding to nucleic acids. Cells were plated on coverslips at a density of 50% confluency in the absence or presence of 5, 15, 25, or 50 µM Roscovitine. In cultures treated with Roscovitine for more than 24 h, fresh medium with the appropriate concentration of the drug was added every 24 h. Coverslips were washed in Dulbecco's phosphate-buffered saline prior to incubation with reagents for the viability assay. As a positive control for dead cells, cells were treated with ice-cold methanol for 10 min and washed with Dulbecco's phosphate-buffered saline. Coverslips were incubated in 150 µl of combined assay reagents (0.5 µM EthD-1 and 0.25 µM calcein AM) for 30 min at room temperature. To ensure minimal damage to the cells, coverslips were mounted on 10 µl of Dulbecco's phosphate-buffered saline on a microscope slide and sealed to prevent evaporation. Green and red fluorescent cells were counted, and the percentage of live and dead cells was determined for mock- and virus-infected cells treated with increasing concentrations of Roscovitine at 24, 48, 72, 96, 120, and 144 h p.i.
Determining the effects of Roscovitine on virus titer. G0-synchronized cells were released from confluence and infected with the Towne strain of HCMV at an MOI of 3 or 5. At the designated times p.i., the indicated concentration of Roscovitine or dimethyl sulfoxide was added to the medium. The medium was changed every 24 h, and fresh drug or control dimethyl sulfoxide was added. Viral supernatants were collected at days 4, 5, and 6, and the titers were determined by plaque assay (65).
Cycloheximide and actinomycin D experiments. Cells were synchronized in G0, trypsinized, and reseeded at a lower density. After allowing the cells to recover for 1 h, the cells were treated for an hour with either 100 µg of cycloheximide (stock solution, 10 mg/ml in distilled H2O) per ml in medium or medium alone prior to infection. Cycloheximide was kept on the treated cells until 6 h p.i. All cells were infected with the Towne virus at an MOI of 3, and 15 µM Roscovitine was added at the time of infection to designated cell cultures. At 6 h p.i., all cells were washed twice with phosphate-buffered saline and fresh medium was added. Roscovitine and actinomycin D (stock solution, 10 mg/ml in methanol) were added to the designated cell cultures to final concentrations of 15 µM and 20 µg/ml, respectively, and the cells were harvested at 18 h p.i.
Experiments with the proteasome inhibitor MG132. G0-synchronized cells were released from confluence and infected with Towne at an MOI of 3 at the time of replating; 15 µM Roscovitine and 2.5 µM MG132 (stock solution, 10 mM in dimethyl sulfoxide) were added to designated plates at the time of infection. At 8 h p.i., all cells were washed twice with phosphate-buffered saline, and fresh medium with the appropriate drugs was added back to the designated cell cultures. Cells were harvested at various times p.i.
Slot blot analysis. DNA was isolated from infected cells at 24, 48, and 72 h p.i. with the Blood and Cell Culture minikit (Qiagen). DNA samples (5, 25, and 125 ng of each sample) were spotted onto 45-µm Nytran (Osmonics) with a slot blot apparatus (Schleicher & Schuell) according to the manufacturer's instructions. The DNA was then cross-linked to the blot with a Stratalinker (Stratagene) as recommended by the manufacturer. The blot was probed with a radiolabeled ApaLI-NcoI fragment of the EcoRI B fragment of HCMV strain AD169 (63) per standard protocols and exposed to autoradiograph film. Band intensity was quantified by measuring the integrated pixel densities as determined with NIH Image and Photoshop 7.0 software. Differences were determined for samples within the linear range.
Western blot analysis. Cells were lysed in Laemmli reducing sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris, pH 6.8, 2 µg each of aprotinin and leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.5 mM Na3VO4, 4 mM EDTA, 10 mM Na4P2O7, 1 mM benzamidine, and 1 mM NaS2O5). The lysates were then sonicated, boiled for 5 min, and centrifuged for 1 min at 16,000 x g. Samples were run on 10% polyacrylamide gels. Proteins were transferred to Immobilon P (Millipore) or Protran (Schleicher & Schuell), and the blots were stained with amido black to ensure that each lane had an equivalent amount of protein. Lysates were also assayed by Western blot for actin. Western blot analysis was performed with the appropriate mouse or rabbit antibody followed by the appropriate horseradish peroxidase-linked secondary antibody (Calbiochem). Proteins were visualized with the West Femto or West Pico (Pierce) detection method (per the manufacturer's instructions). Band intensity was quantified by measuring integrated pixel densities as determined with NIH Image and Photoshop 7.0 software.
Antisera. The antibodies used in Western analysis were anti-IE1 and IE2 (1203; Goodwin Institute), anti-UL112-113 (73), anti-UL44 (1202; Goodwin Institute), anti-UL57 (1209, Goodwin Institute), anti-pp28 (UL99), and anti-major capsid protein (UL86) (gifts from William Britt, University of Alabama), and anti-pp65 (UL83) (1205S; Goodwin Institute).
Northern blot analysis. Total RNA was isolated at 24 h p.i. with the RNAqueous midikit (Ambion). RNA samples (10 µg of each sample) were separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to 0.45-µm Nytran (Osmonics) as previously described (4). Following cross-linking in a Stratalinker (Stratagene), the blot was probed for the UL44 viral transcripts according to standard protocols with the 183-bp EcoRI fragment of the UL44 gene (a gift from Greg Pari, University of Nevada) that was radiolabeled with 32P by random priming. The blot was then exposed to autoradiograph film. Band intensity was quantified by measuring integrated pixel densities as determined with NIH Image and Photoshop 7.0 software.
Quantitative real-time PCR. Cells (107 per sample) were infected with the Towne virus at an MOI of 5, and 15 µM Roscovitine or dimethyl sulfoxide alone was added to the medium at designated times p.i. The cells were harvested at various times p.i., and total RNA was isolated from the harvested cells with a NucleoSpin RNA II purification kit (BD Biosciences Clontech, Palo Alto, Calif.). The concentration of each sample was determined by UV spectrophotometry. Quantitative real-time reverse transcription-PCR was performed in an Applied Biosystems ABI Prism 7700 sequence detection system or in an Applied Biosystems ABI Prism 7000 sequence detection system with the TaqMan One-Step reverse transcription-PCR master mix reagents kit (Applied Biosystems) and oligonucleotide primers and TaqMan dual-labeled (5' fluorescein (FAM) 3' Black hole quencher) probes (Integrated DNA Technologies, Coralville, Iowa) (see Table 1 for sequences).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the cytotoxicity of Roscovitine on mock- and virus-infected HFF cells, we observed the cells for changes in morphology and tested them for viability with the LIVE/DEAD viability/cytotoxicity assay kit (Molecular Probes). Cells were plated on coverslips at a density of 50% confluency and infected with HCMV Towne or mock infected with tissue culture supernatant. At the time of infection, HCMV- and mock-infected cultures were treated with 5, 15, 25, or 50 µM Roscovitine or dimethyl sulfoxide, and cell viability was assayed at 24, 48, 72, 96, 120, and 144 h p.i. In the LIVE/DEAD viability/cytotoxicity assay, calcein dye retained by live cells was observed as green fluorescence, while EthD-1 staining in dead cells was observed as red fluorescence.
During the entire time course, there was no detectable cytotoxicity in mock-infected cultures treated with 5, 15, or 25 µM Roscovitine (<0.1% red cells), although the cultures treated with 15 or 25 µM Roscovitine took longer to reach confluence (data not shown). When treated with 50 µM Roscovitine, the mock-infected cells began to show morphological changes at 72 h postplating, and an increased number of cells exhibited red fluorescence (
1%). This was an underestimate, as the cell density decreased by 50% during the 72 h of incubation, and most of the dead cells were likely removed during the wash steps of the assay. As expected, in the mock-infected culture treated with ice-cold methanol for 10 min prior to the assay, all of the cells were dead and showed bright red fluorescence (data not shown).
We then determined the effects of these increasing concentrations of Roscovitine on the infected cells and viral titers when the drug was added at the time of infection. As shown in Fig. 2, we observed a dose-dependent response in viral titers. The peak titer at day 6 for untreated cultures was 3.8 x 106 PFU/ml. At this time point, cells treated with 5 µM Roscovitine produced approximately the same amount of virus (5.2 x 106 PFU/ml). A greater effect on the infection and viral titer was observed when Roscovitine concentrations of 15 µM and above were tested. We first observed a difference in cytopathic effect at 24 h p.i. in cells treated with these higher concentrations of the drug. At this time, both untreated cells and those treated with 5 µM Roscovitine were completely rounded, an effect of early gene expression, while the cells treated with 15, 25, and 50 µM Roscovitine showed progressively less cytopathic effect.
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In the experiments presented here, we used Roscovitine at a concentration of 15 µM, which did not completely inhibit infection. To determine whether the requirement for the cyclin-dependent kinases in a productive infection was time dependent, we investigated the production of infectious virus when Roscovitine was added at various times during the infection. In the experiment shown in Fig. 3, the titer of wild-type HCMV (Towne) in the absence of Roscovitine at day 6 p.i. was 1.1 x 106 PFU/ml. The addition of 15 µM Roscovitine at the time of infection resulted in a 20-fold reduction in virus production. A similar decrease in viral titer occurred when the drug was added at 12 h p.i. Even when the drug was added at 24 h p.i. (after the onset of viral DNA replication) or at 48 h p.i. (when viral assembly is occurring), virus titers were still reduced by approximately fivefold. In other experiments, a higher level of inhibition was seen at all time points, but the relative difference in inhibition when the drug was added at early and late times was similar. These results suggest that the requirement for cyclin-dependent kinase activity is not restricted to the early phase of the infection.
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Figures 5A and B show that when 15 µM Roscovitine was added at the time of infection, there was a decrease in the amount of IE1-72, while IE2-86 levels increased. At 24 h p.i., the level of IE1-72 protein in untreated cells was approximately two- to threefold higher than in cells treated with the drug. At this time point, there was an increase of approximately 1.5-fold in IE2-86 levels in cells treated with Roscovitine. Coincident with the alterations in viral IE gene expression, viral early gene expression was also affected by the addition of 15 µM Roscovitine. The differential effects on the expression of three genes that play a role in viral DNA replication are shown. The levels of the two major early proteins (43 and 50 kDa) encoded by the UL112-113 locus were only modestly affected by the presence of Roscovitine (Fig. 5A). A greater inhibitory effect was seen on the expression of UL44, the processivity factor for the viral polymerase (Fig. 5A and B). At 24 and 48 h p.i., the levels of UL44 were at least twofold higher in lysates from untreated samples than from Roscovitine-treated cells.
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Delaying addition of Roscovitine until 6 h p.i. abolishes its effects on the steady-state levels of viral proteins. Because the DNA slot blot experiment suggested that there was a window of time within which cyclin-dependent kinase activity was required for viral DNA replication, we asked if this was also true for the effects of Roscovitine on the levels of viral gene products. In the experiment shown in Fig. 6, cells were infected in the absence of Roscovitine or the drug was added at the beginning of the infection or at 6 or 12 h p.i. The infected cells were then harvested at 24 h p.i. In contrast to what was observed when the drug was added at the time of infection, addition of Roscovitine at 6 or 12 h p.i. did not affect the accumulation of IE1-72. The levels of both UL44 and UL57 were partially restored to the levels in the untreated cells by delaying addition of the drug to 6 h p.i. and completely restored by delaying addition of the drug to 12 h p.i.
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The reverse transcription-PCR results for IE1-72 and IE2-86 (Fig. 8A and B) are consistent with the previous Western analysis of IE protein levels. Addition of Roscovitine at the time of infection led to a reduction in IE1-72 mRNA levels and an increase in the levels of IE2-86 mRNA. This was particularly evident at the 8-h time point.
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Roscovitine affects the differential processing and accumulation of the IE UL37 RNAs. The major UL37 IE promoter directs the synthesis of several spliced 3.2- to 3.4-kb RNAs (UL37 and UL37M) that are present in small amounts only at IE times. It is also the promoter for an abundant 1.7-kb unspliced RNA (UL37X1) that is present throughout the infection (see Fig. 1C). Examination of the relative position of the sequences used for processing the HCMV unspliced and spliced UL37 IE RNAs (64) revealed a striking similarity to the region between UL123 exon 4 and UL122 exon 5 that is used for the alternative cleavage-polyadenylation and splicing that generates the IE1-72 and IE2-86 RNAs (Fig. 9A). The cleavage-polyadenylation site for the IE1-72 transcript (a binding site for the cleavage factors CFI and CFII) lies just upstream (33 nucleotides) of the splice acceptor site for the terminal exon of IE2. It also lies within a consensus mammalian splice branch point (UACUAAG).
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Samples of RNA used for the quantitative real-time PCR analysis shown in Fig. 8 were assayed with primers and probes specific for the unspliced UL37X1 and the spliced UL37 mRNAs (Table 1). Figure 9 shows that addition of Roscovitine decreased the mRNA levels of the unspliced UL37X1 transcript (Fig. 9B) and at the same time increased the amount of spliced UL37 mRNA (Fig. 9C). Thus, Roscovitine had the same effect on the processing and accumulation of the UL37 RNA as it did on the UL122-123 RNA.
Effects of Roscovitine on IE gene expression occur in the absence of de novo protein synthesis. In order to determine if the effects of Roscovitine on IE1-72 and IE2-86 expression required new protein synthesis, we infected cells in the presence of cycloheximide, Roscovitine, cycloheximide plus Roscovitine, or medium alone (Fig. 10). In the presence of cycloheximide, no host or viral proteins are synthesized, and therefore only the IE RNAs are transcribed. At 6 h p.i., the cycloheximide was removed so that protein synthesis could resume. Cells that were infected in the presence of cycloheximide alone (Fig. 10, lanes 3 and 6) were refed with medium alone (Fig. 10, lane 3) or medium plus actinomycin D to inhibit further transcription of IE and early RNAs (Fig. 10, lane 6). Cells that had been infected in the presence of Roscovitine alone (Fig. 10, lanes 2 and 7) or Roscovitine plus cycloheximide (Fig. 10, lanes 5 and 8) were refed with medium containing Roscovitine (Fig. 10, lanes 2 and 5) or with Roscovitine plus actinomycin D (Fig. 10, lanes 7 and 8). As a control, cells that were infected in the absence of both cycloheximide and Roscovitine (Fig. 10, lanes 1 and 4) were refed with medium alone (Fig. 10, lane 1) or with medium plus actinomycin D (Fig. 10, lane 4). All cells were harvested at 18 h p.i. and analyzed by Western blot for the expression of the IE1-72 and IE2-86 proteins.
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Effects of Roscovitine on IE gene expression are proteasome independent. An alternative explanation for the altered transcriptional processing was that Roscovitine affected the ubiquitin proteasome degradation pathway such that a positive factor for the processing of the IE1-72 RNA was rapidly degraded. If this were the case, then inhibiting the proteasome to prevent protein degradation should allow IE1-72 protein levels to be restored. To test this hypothesis, we infected cells in the presence of Roscovitine alone, MG132 alone (a proteasome inhibitor), Roscovitine plus MG132, or medium. The cells were harvested at 8, 16, and 24 h p.i. and analyzed by Western blot for IE1-72 and IE2-86 gene expression. Figure 11 shows that inhibition of the proteasome did not have an effect on the levels of the IE proteins in the absence of Roscovitine and did not alter the Roscovitine-mediated change in the relative amounts of IE1-72 and IE2-86. Based on these results, we conclude that Roscovitine is not causing the rapid proteasome-mediated turnover of a factor needed for IE1-72 gene expression.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In accord with those of others (9, 24), our experiments showed that addition of Roscovitine to the medium at the beginning of the infection significantly reduced HCMV titers. Viral DNA replication was also inhibited; however, both of these phenomena could have been due to the lack of viral gene expression. Given that HCMV infection proceeds in a temporal fashion, where one event is required before the next can occur, the loss of viral gene expression would lead to the loss of replication and in turn the loss of virus production.
When Roscovitine was added at the beginning of the infection, the amount of the IE1-72 protein decreased, while the levels of IE2-86 increased. Because IE1-72 is a known transactivator of early gene expression, it was not surprising that the levels of some early gene products (UL44 and UL57) were also reduced. Along similar lines, because UL44 and UL57 are required for HCMV replication (51), it followed that viral DNA replication would also be affected. With immunofluorescence analysis, we noted that replication centers formed but never grew and coalesced in the presence of Roscovitine (data not shown). Morphologically, the replication centers in Roscovitine-treated cells at late times of infection were similar in appearance to the replication centers that are seen very early in untreated cells.
Importantly, it is unlikely that the effects of Roscovitine are due entirely to decreased levels of IE1-72, as it has been shown that an IE1-72 deletion mutant can replicate efficiently at a high MOI. Since all of the experiments reported in this paper were conducted at a high MOI, it is almost certain that other events are occurring early during the infection that are cyclin-dependent kinase dependent. Most of these events likely occur prior to 6 h p.i., because a 6-h delay in the addition of Roscovitine to the infected cells allowed viral gene expression to progress normally to the late phase, although viral titers were still affected. These results suggest that although cyclin-dependent kinase activity is not directly required for viral DNA replication, it is required at later times, possibly for virion assembly.
The early effect on IE1-72 and IE2-86 gene expression appears to be at the level of processing of the RNA. During the first 24 h of the normal infection, IE1-72 RNA accumulates to a higher level than IE2-86 RNA. As time progresses, more IE2-86 transcripts accumulate and there is a slight increase in IE1-72 transcripts. However, when Roscovitine was present during the first 24 h of the infection, the ratio of IE1-72 RNA to IE2-86 RNA was reversed; there was a decrease in IE1-72 RNA and an increase in the level of the IE2-86 transcript. The most likely explanation for these data is that the differential splicing of the UL122-123 transcript was altered by Roscovitine treatment, but we cannot exclude the possibility that there was a change in the relative stability of the RNAs. Treatment with the drug appeared to affect the differential splicing and polyadenylation of the IE UL37 RNAs in a similar way.
We hypothesize that for both the UL122-123 and UL37 transcripts, Roscovitine and Flavopiridol treatment leads to suppression of the first cleavage-polyadenylation site and enhanced utilization of the adjacent downstream 3' splice acceptor site. Relevant to this are the recent studies of Su et al. (64). Although their experiments used mutant target minigenes and transient transfection assays in uninfected cells, the results indicated that the cis polyadenylation and 3' splice acceptor sites for the UL37 RNAs caused steric hindrance on the precursor mRNA for the alternative processes of polyadenylation and splicing, with polyadenylation of UL37X1 being dominant. In the case of the UL122-123 transcripts, the polyadenylation site generating the IE1-72 RNA is also juxtaposed to the 3' splice acceptor site that is used to produce the IE2-86 RNA. In this case, the U-rich polypyrimidine sequence upstream of the 3' splice acceptor could provide a binding site for the cleavage-polyadenylation complex CstF, the splicing suppressor polypyrimidine tract binding protein, or the essential U2AF-65 splicing factor (Fig. 9).
Likewise, the sequence where the cleavage occurs at the 3' end of the IE1-72 RNA could be used for the binding of the cleavage factors CFI/II. However, this sequence also lies within the sequence UACUAAG, which matches the mammalian splicing branch point consensus sequence (YNYURAY) except for the last position, which can be changed without a significant decrease in activity (53), and thus it could serve as the site for the binding of SF1 and U2 splicing factors. Su et al. additionally reported that during HCMV infection, there was a transient increase in CstF64, the splicing suppressor polypyrimidine tract binding protein, and the hypophosphorylated form of the splicing factor SF2 at 4 h p.i. We also examined the expression of these factors but did not detect these changes during the first 8 h p.i. even when the infected cells were analyzed every hour (data not shown). Their expression was also not affected by treatment of the cells with Roscovitine. The reason for the discrepancy is not clear but may be due to the use of different strains of HCMV (AD169 in the studies of Su et al. versus Towne in our studies) or the state of the fibroblasts (unsynchronized versus synchronized).
There is a large body of evidence showing that the processes of mRNA capping, splicing, and cleavage-polyadenylation occur cotranscriptionally and are highly interdependent. Given the proximity of the polyadenylation site and the splice acceptor site, it seems likely that there is competition for sites on the RNA that may be required for both processes. Since numerous factors are involved in both splicing and cleavage-polyadenylation, a change in the abundance, activity, or localization of any of the factors could alter the balance. The phosphorylation state of many of the factors involved in both processes determines their activity, and therefore the cyclin-dependent kinases may play a direct or indirect role in their phosphorylation. Because multiple negative and positive splicing factors are differentially phosphorylated, it is difficult to predict whether inhibition of the cyclin-dependent kinases results in suppression of the cleavage-polyadenylation of IE1-72 and UL37X1 by the loss of activity of a positive factor or gain of activity of a negative factor. It remains to be seen which cyclin-dependent kinase might be the important one for the differential processing of the UL122-123 and UL37 transcripts and which specific proteins are important for this level of regulation of viral gene expression. Based on the results of the experiments with cycloheximide and MG132, it appears that the effects of Roscovitine on the IE RNAs do not require de novo protein synthesis or proteasome-mediated degradation of proteins.
It is also possible that the effects of Roscovitine and Flavopiridol are related to the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II. There is increasing evidence that the C-terminal domain, which in human cells consists of 52 repeats of the consensus heptapeptide sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, plays a central regulatory role in all steps of transcription by serving as the binding domain and transporter of factors involved in RNA initiation, elongation, 5' capping, splicing, and cleavage-polyadenylation (for review, see references 5, 48, and 57). The C-terminal domain is differentially phosphorylated primarily at the serine 2 and serine 5 positions, and the level of phosphorylation varies considerably during the transcription cycle (for review, see reference 39 and 55).
Briefly, hypophosphorylated-RNA polymerase II is recruited to the initiation complex. The C-terminal domain is then phosphorylated, with the phosphorylation of the serines in position 5 by cdk7/cyclin H (which is a component of the basal transcription factor complex TFIIH) mostly occurring prior to the phosphorylation of the serines in position 2 by cdk9/cyclin T (P-TEFb). The commitment of the RNA polymerase II complex to the elongation step is associated with this phosphorylation, particularly with the modification at position 2 by P-TEFb. Multiple proteins involved in 5' capping, elongation, and processing of the RNA are recruited to the C-terminal domain, and their pattern of binding appears to be influenced by the differential phosphorylation of serine 2 and serine 5 (and possibly by ubiquitylation, glycosylation, and phosphorylation of other residues) within the 52 repeats. The rules governing this association in uninfected cells have yet to be elucidated, and it is likely that viral infections will introduce new layers to the complexity.
In this regard, several studies have already shown that the recruitment of P-TEFb to RNA polymerase II by human immunodeficiency virus type 1 Tat and the resulting phosphorylation of the C-terminal domain are necessary for the activation of human immunodeficiency virus long terminal repeat transcription (for review, see reference 56). Human immunodeficiency virus type 1 transcription is much more sensitive than cellular transcription to low concentrations of Flavopiridol; the IC50 for human immunodeficiency virus transcription is less than 10 nM, while significant inhibition of cellular transcription is only seen at concentrations above 100 nM (13). In herpes simplex virus type 1-infected cells, a new intermediate form of phosphorylated RNA polymerase II has been described (32, 58). Although preliminary, we also have evidence that there is a change in the phosphorylation of the C-terminal domain during HCMV infection, but it appears to differ from that reported for herpes simplex virus type 1.
The question of why a delay in the addition of Roscovitine and Flavopiridol until 6 to 8 h p.i. abolished the effects of the drug on IE and early RNAs is an interesting one. It is possible that by this time, transcription of the HCMV genome is localized to a nuclear domain (possibly next to a residual ND10 domain) that is inaccessible to the drug. Alternatively, once the RNA polymerase II is committed to the transcription of the viral genes, it might be able to engage in multiple rounds of transcription without having to completely disengage from the DNA and repeat all of the initiation steps. Relevant to this are recent reports showing that there are many protein-protein interactions between the factors involved in initiation at the promoter and termination of transcription, suggesting that these two processes are interconnected (for review, see reference 48).
These are only a few of the possible ways that the cyclin-dependent kinases might affect infection. Unfortunately, the use of inhibitors such as Roscovitine and Flavopiridol, which inhibit more than one cyclin-dependent kinase, makes it difficult to distinguish between these possibilities. There are likely many more functions for cyclin-dependent kinase activity than transcription during the course of an HCMV infection, and this is evidenced by the fact that addition of Roscovitine at later times still resulted in a decrease in viral titers. Another potential role for cyclin-dependent kinase activity during an HCMV infection is the phosphorylation of viral proteins. In a related virus, herpes simplex virus type 1, it was demonstrated that UL42 (the herpes simplex virus equivalent of UL44) bound to and was phosphorylated by cdk1 (1). The effects that this may have on viral replication and the potential downstream targets of this kinase complex are unknown; however, the implications of these data as they pertain to HCMV infection are promising. Experiments with dominant negative cyclin-dependent kinases and the technology of small interfering RNAs coupled with analysis of the splicing machinery and of viral targets of cyclin-dependent kinase activity are in progress and aim to elucidate the role of the individual cyclin-dependent kinases during infection.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants CA73490 and CA34729 to D.H.S. A.K.M. was supported by NIH training grant CA09345. J.Y.Y. was supported by NIH training grant GM07240.
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FOOTNOTES |
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Present address: The Salk Institute for Biological Studies, La Jolla, CA 92037.
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