Department of Public Health and Microbiology, University of Torino, Via Santena, 9 10126 Torino, Italy1
Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA2
Author for correspondence: Giorgio Gribaudo. Fax +39 011 6636436. e-mail giorgio.gribaudo{at}unito.it
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Abstract |
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Introduction |
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TS is an essential enzyme that catalyses the de novo biosynthesis of thymidylic acid (dTMP) by the reductive transfer of the methylene group from 5,10-methylenetetrahydrofolate to the 5 position of the substrate, deoxyuridylic acid, to form dTMP and dihydrofolate. TS activity is associated with cell proliferation and its inhibition in rapidly proliferating cells leads to inhibition of DNA synthesis and cell death. For this reason, TS is an important target enzyme in cancer chemotherapy. Both substrate and cofactor analogues have been widely used as anti-neoplastic agents. TS mRNA and enzyme levels are very low in quiescent cells, but increase at the G1S border during a serum-induced transition from the resting (G0) phase to the S phase (Jenh et al., 1985 ; Ayusawa et al., 1986
). TS mRNA content is primarily controlled at the post-transcriptional level in growth-stimulated cells, since nuclear run-on transcription assays have revealed that TS gene transcription in human and mouse cells does not change during the G1S transition (Ayusawa et al., 1986
; Ash et al., 1995
). Studies with transfected TS minigenes have shown that both the TS essential promoter region and an intron in the transcribed region are required for proper S-phase regulation, suggesting that some form of communication between the TS promoter and the RNA processing machinery may be important for regulation of TS mRNA production in growth-stimulated cells (Kaneda et al., 1992
; Takayanagi et al., 1992
; Ash et al., 1993
, 1995
; Johnson, 1994
; Ke et al., 1996
).
We have been studying the effects of CMV infection on the expression of enzymes involved in dTMP biosynthesis, since elucidation of the molecular mechanisms of virus-mediated regulation could lead to the design of molecules with antiviral activity. In the murine system, we have observed that murine CMV (MCMV) replication in quiescent NIH 3T3 cells increases the expression of enzymes involved in dTMP biosynthesis, namely folylpolyglutamate synthetase (FPGS), DHFR and TS (Lembo et al., 1998 ; Gribaudo et al., 2000
; Cavallo et al., 2001
). Here we report that HCMV infection of quiescent human embryonic lung fibroblasts induces the expression of TS mRNA and protein, and that TS activity is required for efficient HCMV DNA synthesis. Regulation of TS gene expression takes place at the transcriptional level, and several DNA elements within the TS promoter are necessary for its increase. Lastly, HCMV and MCMV transactivate the corresponding cellular TS promoters by different mechanisms.
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Methods |
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Virus preparation and infections.
HCMV (strain AD169; ATCC VR-538) was passaged in HELF cells. MCMV (strain Smith; ATCC VR-194) was propagated in NIH 3T3 cells. Both viruses were prepared in low-serum medium.
Quiescent HELF or NIH 3T3 cells were infected with HCMV or MCMV, respectively, at an m.o.i. of 5, unless otherwise stated. Mock-infected control cultures were exposed to an equal volume of mock-infecting fluid. At the end of the adsorption period, which was defined as 0 h post-infection (p.i.), the low-serum medium removed before infection was restored to the cultures to avoid stimulation due to the addition of fresh serum growth factors. UV-inactivated HCMV was prepared with one pulse of 1·2 J/cm2 UV light. The UV-inactivated HCMV did not replicate or produce detectable levels of immediate-early (IE) gene products.
For infection in the presence of cycloheximide (CHX), quiescent HELF cells were pretreated for 1 h with 100 µg/ml CHX before infection and then maintained in the medium throughout the infection. For infection in the presence of phosphonoformic acid (PFA), PFA was added to the medium at a final concentration of 200 µg/ml at the time of infection. RNA was then harvested at 24 (CHX) or 48 (PFA) h p.i.
Northern blot analysis.
Total RNA or poly(A)+ mRNA was fractionated on a 1% agarose, 2·2 M formaldehyde gel and then blotted on to a nitrocellulose membrane (Hybond C-extra; Amersham). The filters were hybridized to random-primed radiolabelled probes corresponding to the human TS cDNA (Ayusawa et al., 1984 ) and human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA as a control.
Plasmids.
Plasmids containing the indicated regions of the human TS promoter (numbered relative to the A of the translational start codon; see Fig. 3) driving expression of the luciferase indicator gene were constructed in the following manner. Plasmids phTS -161/-98 Luc, phTS -161/-141 Luc and phTS -140/-98 Luc were constructed by inserting the HindIIINheI fragment of the corresponding TS minigene (Dong et al., 2000 ) into the HindIII and NheI sites of pGL3 basic-EP, which is the same as pGL3 basic (Promega) except that the multiple cloning sequence was modified by the insertion of a synthetic DNA fragment to give a new multiple cloning sequence (KpnI, HindIII, SacI, XbaI, NheI, SmaI, BglII). Plasmid phTS-161/-141 Sp1 mut. Luc was constructed by inserting synthetic oligonucleotides with the desired nucleotide changes into the HindIII and NheI sites of pGL3 basic-EP. Plasmid phTS -243/+30 Luc was constructed by PCR-amplifying the indicated region of the human TS promoter and inserting it into the NheI site of pGL3-basic that had been modified by deleting 13 nucleotides between the BglII and HindIII sites (Dong et al., 2000
). The promoter regions of all constructs were sequenced to verify the presence of the desired promoter regions and/or mutations and the absence of undesired changes. Plasmids phTS -243/-70 Luc, phTS -243/-98 Luc and phTS -243/-141 Luc were as described previously (Dong et al., 2000
). pTSWTGL3 was constructed by inserting the mouse TS promoter and 5' flanking region from the XbaI site at -985 to an engineered BglII site at -11 into the NheI and BglII sites of pGL3-basic. pTSWTGL3(-110) was the same as pTSWTGL3, except that the potential E2F element just upstream from the mouse TS essential promoter region (-115GATTCTGGCGGCC-103) was mutated to -115GATTCGCTAGCCC-103 (Geng & Johnson, 1993
). pSGIE72 and pSGIE86 contained cDNAs corresponding to the HCMV IE1 72 kDa and IE2 86 kDa proteins, respectively. Their expression was driven by the simian virus 40 (SV40) early promoter (Klucher et al., 1993
). p729CAT contained the CAT reporter gene driven by the HCMV UL112/113 early promoter (Staprans et al., 1988
).
Transient transfection and reporter gene assays.
Cells were transfected by the calcium phosphate procedure, as previously described (Gribaudo et al., 2000 ). The transfected cells were washed twice with medium and incubated in MEM supplemented with 0·5% foetal calf serum (low-serum medium) for 48 h. Luciferase and CAT activity were assayed as previously described (Gribaudo et al., 1995
). Reporter gene activity was normalized to the amount of plasmid DNA introduced into recipient cells, as previously described (Abken & Reifenrath, 1992
).
Immunoblotting.
Whole-cell protein extracts were prepared as previously described (Gribaudo et al., 2000 ). Proteins were separated by SDSPAGE and then transferred to Immobilon-P membranes (Millipore). Filters were immunostained with the mouse anti-TS human mAb (Johnston et al., 1991
) (clone TS106; Labvision Neomarkers), the mouse anti-HCMV IEA mAb (E13 clone; Argene Bio-Soft) recognizing both the IE1 72 kDa and IE2 86 kDa proteins, or the mouse anti-actin mAb (Boehringer) at room temperature for 1 h. Immune complexes were detected with sheep anti-mouse IgG antibody conjugated to horseradish peroxidase (Amersham) and visualized by enhanced chemiluminescence (Super Signal; Pierce).
Inhibition of viral DNA synthesis.
To evaluate the inhibition of HCMV DNA synthesis, cells were grown to subconfluence, incubated in low-serum medium for 48 h and infected with HCMV at an m.o.i. of 1. The infected cultures were treated in low-serum medium with different concentrations of N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (raltitrexed, Tomudex, formerly ZD1694; Zeneca). At 96 h p.i. cells were harvested and total DNA was isolated. Dot-blot hybridization was then performed using 32P-labelled probes prepared from the PstIBamHI DNA fragment of the HCMV IE1 gene (exon 4) and mouse G3PDH cDNA. The membranes were autoradiographed and the hybridization signals were quantified using a phosphoimager.
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Results |
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To determine whether viral infection would also lead to an increase in TS protein, cell extracts were prepared at different times p.i. and analysed by immunoblotting with an anti-TS mAb. As shown in Fig. 2(C), TS protein was undetectable in mock-infected cells, started to increase at 24 h p.i. in infected cells and peaked at 72 h p.i. In contrast, it was undetectable in cells infected for 48 h with UV-inactivated HCMV, further demonstrating that newly synthesized viral proteins are required to induce cellular TS expression. The efficacy of UV treatment was confirmed by the lack of viral IE gene expression. These results confirmed that the increases in TS mRNA and protein levels during HCMV replication in quiescent HELF cells depend on viral gene expression.
HCMV infection transactivates the TS promoter in quiescent HELF cells
To determine whether the increase in TS mRNA was a consequence of stimulation of TS promoter activity, we analysed the effects of HCMV infection on the expression of a transiently transfected luciferase reporter gene driven by the human TS promoter. HELF cells were transiently transfected with the indicator plasmid phTS -243/+30 Luc, which corresponds to an intronless luciferase gene driven by the promoter and adjacent sequences of the human TS gene (Fig. 3). These promoter sequences are sufficient to drive high-level expression of an indicator gene (Dong et al., 2000
). Transfected cells were serum-starved and then infected with HCMV or UV-inactivated virus, mock-infected or stimulated with serum. At different times p.i., cells extracts were prepared and assayed for luciferase activity. As shown in Fig. 4
, HCMV infection led to a sixfold increase in luciferase activity by 12 h, a 26-fold increase by 24 h and an 18-fold increase by 48 h p.i. UV-inactivated virus did not affect luciferase activity, demonstrating that HCMV-mediated transactivation requires de novo viral protein expression. As expected, serum stimulation did not increase the reporter activity since, as previously observed, the S-phase-specific human TS expression requires the presence of both the promoter region and a spliceable intron (Takayanagi et al., 1992
). Taken together, these results demonstrate that HCMV regulates TS gene expression primarily at the transcriptional level using mechanisms that are different from those employed by growth-stimulated cells.
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We have recently observed that MCMV infection transactivates the mouse TS promoter and that this transactivation was abolished by inactivating the functional E2F element immediately upstream of the mouse TS essential promoter region. These observations indicated an E2F-dependent mechanism for induction of the mouse TS promoter in response to MCMV infection (Gribaudo et al., 2000 ). The presence of two potential E2F binding sites within the inverted repeat element between -128 and -98 of the human TS promoter (Fig. 1
) raised the possibility that these elements may contribute to the regulation of human TS promoter activity in response to HCMV infection. We recently found that recombinant E2F is able to bind to the human TS promoter region between -134 and -105 in EMSA assays (data not shown). Unexpectedly, we found that the E2F elements were not necessary for the HCMV-mediated increase in TS promoter activity since a similar increase in luciferase activity was observed with phTS -161/-141 Luc, which lacks the E2F elements, as with phTS -161/-98 Luc, which retains the E2F elements (Fig. 5
).
Taken together, these results indicate that the -98/-70 element and the minimal essential TS promoter segment play important positive roles in the response of the human TS promoter to HCMV infection. Furthermore, in contrast to our earlier observations in the murine system, stimulation of the human TS promoter by HCMV does not appear to involve an E2F-dependent mechanism.
HCMV IE1 protein transactivates the human TS promoter
We have previously observed that the expression of MCMV IE1 protein activates the mouse TS promoter and that this activation is dependent on the integrity of the E2F element (Gribaudo et al., 2000 ). To examine the potential role of the HCMV major immediate-early proteins in the regulation of the human TS promoter, we co-transfected an expression vector for the IE1 72 kDa or the IE2 86 kDa protein with different human TS constructs into HELF cells. The major HCMV IE proteins were expressed under the control of the early SV40 promoter to avoid the potential complication of negative autoregulation of the HCMV IE promoters by the IE2 protein (Klucher et al., 1993
). Fig. 6
demonstrates that only the IE1 product transactivated the human TS promoter, although to a lesser extent than that observed with virus infection. The magnitude of activation was 4·5-fold for phTS -243/+30 Luc, 3·5-fold for phTS -243/-70 Luc, threefold for phTS -243/-98 Luc and 2·5-fold for phTS -243/-141 Luc relative to the control. None of the other constructs analysed was significantly transactivated by IE1 (data not shown).
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Differential regulation of mouse and human TS promoters by CMV infection
As indicated above, activation of the mouse TS promoter by MCMV is mediated through the E2F site in the mouse TS promoter (Gribaudo et al., 2000 ), whereas activation of the human TS promoter by HCMV does not depend on the presence of the E2F sites. This discrepancy prompted us to see whether HCMV regulates the mouse TS promoter by a different mechanism than that used by MCMV. We reasoned that if HCMV were able to control the mouse TS promoter through the E2F element, differences in the architecture of the regulatory elements between the human and mouse TS promoter might account for the different observations. However, if the mouse TS promoter were not regulated in the same manner, this would indicate that the two viruses exploit different molecular mechanisms.
To explore these possibilities, HELF cells were transiently transfected with the pTSWTGL3 and pTSWTGL3(-110) constructs, serum-starved and then infected with HCMV for 24 h. The pTSWTGL3 contains the luciferase gene driven by the wild-type promoter and 5' flanking region of the mouse TS gene. pTSWTGL3(-110) is the same as pTSWTGL3 except that the E2F binding site at -110 (TCTGGCGG) has been mutated to TCGCTAGC (Geng & Johnson, 1993 ). Fig. 7(A)
shows that pTSWTGL3 and pTSWTGL3(-110) were transactivated by HCMV infection by fivefold and by more than 11-fold respectively. Surprisingly, inactivation of the E2F binding site led to a more pronounced response to HCMV infection than that measured with the wild-type construct.
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Discussion |
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The dependence of HCMV replication on TS activity is supported by the results obtained with the folate analogue raltitrexed, a powerful TS inhibitor that prevents DNA synthesis and repair by blocking the de novo synthesis of dTMP. We have shown that raltitrexed inhibited HCMV DNA synthesis in quiescent cells, indicating that induction of TS activity is required for viral DNA replication. In line with this, we have previously observed that raltitrexed had little effect on MCMV and HCMV IE gene expression, although it strongly inhibited late gene expression as well as the replication of the virus in quiescent cells (Lembo et al., 2000 ). Taken together, these observations suggest that raltitrexed inhibits HCMV replication by blocking viral DNA synthesis. We also found that raltitrexed is far more detrimental to CMV DNA replication than to the survival of uninfected quiescent cells. This is probably due to the fact that TS is present at very low levels and is irrelevant for the survival of quiescent cells (Jenh et al., 1985
; Johnson, 1994
), but is required for viral DNA replication. These observations suggest that drugs that target TS may be highly effective in treating CMV infections.
Several laboratories have recently shown that HCMV infection blocks cell-cycle progression and cellular DNA replication while activating the cellular DNA synthetic machinery. These alterations create a favourable environment for high-level viral DNA replication, since they provide the viral DNA polymerase with dNTPs while preventing competition by cellular DNA synthesis (Fortunato et al., 2000 ; Flemington, 2001
). Activation of host genes important for DNA synthesis has been reported to depend on binding of CMV to the cell surface or expression of viral IE proteins (Fortunato et al., 2000
). Here we have shown that: (i) TS mRNA upregulation was prevented in the absence of protein synthesis; and (ii) inactivation of HCMV by UV exposure abolished both the induction of TS protein and the activation of the TS promoter. These results indicate that viral IE and E gene expression, rather than interaction of viral particles with the cell surface, is required to stimulate TS gene expression. Furthermore, we have shown that the product of the IE1 gene partially transactivated the TS promoter and that the virus-dependent activation of TS transcription coincided with expression of the IE1 protein. However, the difference between the levels of transactivation of the human TS promoter in HCMV-infected and in IE1-transfected cells suggests that viral IE and/or E gene products other than IE1 contribute to the overall response of the human TS promoter to virus infection.
To map the sequences important for activation of the TS promoter by HCMV, we assessed its ability to stimulate TS promoters modified by deletions or point mutations. Two positive-acting elements were identified. The first was located between -70 and -98 (relative to the AUG start codon) and corresponded to one of the direct repeats of a 28-nucleotide G/C-rich sequence. The human TS promoter is polymorphic, containing either two or three tandem direct repeats in addition to a single inverted copy of the repeat (Horie et al., 1995 ; Marsh et al., 1999
). The promoter examined in this study had two copies of the direct repeat sequence. We showed that deletion of the downstream direct repeat had little effect on TS promoter activity following HCMV infection, but elimination of both direct repeats significantly reduces expression of the reporter gene in virus-infected cells. Earlier studies did not identify potential binding sites for transcription factors within the repeated sequences (Horie et al., 1992
). In addition, we have shown that deletion of all of the direct repeats has little effect on expression of reporter genes driven by the TS promoter in proliferating cells (Dong et al., 2000
). Therefore, the direct repeat may contribute to the human TS gene expression only under certain conditions, such as virus infection. The repeated sequences have also been reported to affect the efficiency of translation of TS mRNA (Kaneda et al., 1987
; Horie et al., 1995
; Kawakami et al., 2001
). For this reason, it is not clear if the repeated sequence has a positive effect on TS promoter activity or on mRNA translation following HCMV infection.
The second postive-acting element was located between -161 and -141. This segment has been defined as the TS essential promoter region, since it is both necessary and sufficient for efficient promoter activity in proliferating cells. This region contains binding sites for Ets, Sp1 and LSF transcription factors, and inactivation of any of these motifs leads to a significant decrease in promoter activity (Dong et al., 2000 ; Powell et al., 2000
). Previously, it was shown that Sp1 binds to its cognate motif within the essential region of the human TS promoter (Horie & Takeishi, 1997
) and that an increase in Sp1 DNA binding activity occurs in HELF cells during HCMV infection (Yurochko et al., 1995
, 1997
). However, mutation of the Sp1 site (Fig. 3
) in the phTS -161/-141 Luc construct had no significant effect on stimulation of TS promoter activity following virus infection (data not shown), suggesting that the Sp1 site is not important for the response to HCMV infection. It remains to be established whether the Ets and/or the LSF elements may be required for the stimulation of the TS essential promoter region by HCMV. Relevant to this, the DNA-binding activity of an Ets family member, Elk-1, was found to increase in HCMV-infected fibroblasts (Chen & Stinski, 2000
).
The human TS promoter has two E2F-binding sites within the inverted repeat sequence downstream from the essential promoter region (Dong et al., 2000 ). However, deletion of both of these E2F motifs did not significantly diminish the expression of the reporter gene following HCMV infection, indicating that these sequences do not have a significant role in the response of the TS promoter to the virus. This is surprising since it has been reported that HCMV infection results in an E2F-dependent activation of the DHFR promoter (Wade et al., 1992
; Margolis et al., 1995
) and since an E2F element is necessary for the activation of the mouse TS promoter by murine CMV (Gribaudo et al., 2000
).
These observations raised the possibility that MCMV and HCMV may activate the TS promoter by different mechanisms. To explore this possibility, we examined the effect of MCMV and HCMV on the expression of the human or mouse TS promoters, respectively. We found that the murine TS promoter with a mutated E2F site was activated to a higher extent than the wild-type promoter when introduced into human cells that were then infected with HCMV. In contrast, deletion of both E2F sites of the human TS promoter abolished its response to MCMV when the construct was introduced into NIH 3T3 cells. These results suggest that MCMV activates the TS promoter through an E2F-dependent pathway, whereas HCMV relies on a different mechanism.
TS catalyses the de novo synthesis of dTMP by the reductive methylation of dUMP, and availability of the substrate may thus be rate-limiting for dTMP biosynthesis. There are several pathways by which dUMP can be produced in the cell, although deamination of dCMP by deoxycytidylate deaminase (dCMP deaminase) appears to be the most important route. dCMP deaminase activity is much higher in rapidly dividing cells than in quiescent cells and is expressed at the highest levels during the S phase of the cell cycle (Maley & Maley, 1990 ). We have recently observed that HCMV infection of quiescent HELF cells also leads to an increase in dCMP deaminase gene expression (data not shown). These findings, along with previous studies showing increased DHFR activity during HCMV infection (Lembo et al., 1999
), demonstrate that HCMV is able to coordinately activate the expression of multiple cellular enzymes involved in the synthesis of dTMP, thereby releasing the virus from the requirement of an S-phase environment for its replication.
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Acknowledgments |
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References |
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Received 7 February 2002;
accepted 22 August 2002.