The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-κB Sites in Proliferating Cells^▿

Oligonucleotides.

All oligonucleotides used for PCR, mutagenesis, or sequencing were obtained from Invitrogen (Table 1 shows all oligonucleotide sequences).

Cells and culture conditions.

Low-passage human embryonic lung fibroblasts (HELFs) were grown as monolayers in minimum essential medium (MEM) (Gibco-BRL) supplemented with 10% fetal calf serum (FCS) (Gibco-BRL), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate (high-serum medium). Quiescent HELFs (arrested in G₀/G₁ phase) were obtained by culturing subconfluent cultures for 96 h in minimum essential medium (MEM) containing 0.5% FCS (low-serum medium). Flow cytometry demonstrated that more than 90% of the cells were growth arrested.

Virus preparation and infections.

HCMV strain AD169 (VR-538) was purchased from the ATCC. Viral stocks were prepared by infecting HELF cells at a virus-to-cell ratio of 0.01. Cells were incubated in MEM supplemented with 1% heat-inactivated FCS and cultured until a marked cytopathic effect was observed. Stocks then were prepared from sonicated cells, subjected to centrifugal clarification, and frozen at −80°C. Mock infection fluid was prepared from uninfected cells by the same procedure. Virus titers were determined by plaque assay on HELFs. For the determination of viral replication kinetics, quiescent or proliferating HELFs were infected with fusion-inducing factor X (FIX)-bacterial artificial chromosome (BAC)-derived viruses at a multiplicity of infection (MOI) of 0.1. Mock-infected control cultures were exposed to an equal volume of mock-infecting fluid. Virus adsorptions were carried out for 2 h at 37°C. For all experiments, the time at which virus first was added to the cells is considered time zero. Following infection, cultures were maintained in low-serum medium or 10% serum medium for various times postinfection (p.i.). Thereafter, the cells and supernatants were harvested and disrupted by sonication. Viral titers then were measured by an indirect immunoperoxidase staining procedure on HELF cells (13). The HCMV strain used in the control experiments for the growth kinetics analysis was reconstituted from the FIX-BAC containing the genome of HCMV clinical isolate VR1814 (16, 32).

BAC mutagenesis.

The HCMV IE enhancer sequence (positions −52 to −667 relative to the IE1/IE2 transcription start site) was amplified out of the purified HCMV VR1814 genome by PCR using the primers shown in Table 1. The HCMV VR1814 IE enhancer fragment then was ligated between restriction sites HindIII and NheI of the pGL3-basic vector (Promega) to obtain the pMIEPGL3 construct. The correctness of the amplified viral sequences was confirmed by sequencing. To derive MIEP sequences with inactivated binding sites for AP-1, Elk-1, SRF, SEE (Elk-1 and SRF), and all four NF-κB sites (designated 4NF-κB), a Stratagene QuikChange XL site-directed mutagenesis kit was used. The AP-1 (at −169 with respect to the IE1/IE2 transcription start site), Elk-1 (at −522), SRF (at −532), SEE (Elk-1 plus SRF; from −521 to −539), and four NF-κB (at −98, −161, −265, and −412) sites of pMIEPGL3 were changed to unique restriction sites (−169 to −174, EcoRI; −522 to −528, EcoRI; −532 to −538, KpnI; −98 to −103, StuI; −161 to −166, −265 to −270, and −412 to −421, BglII) using the oligonucleotides AP-1-mut-EcoRI, Elk-1-mut-EcoRI, SRF-mut-KpnI, SEE-mut-EcoRI-KpnI, NF-κB-1-mut-StuI, NF-κB-2-mut-KpnI, NF-κB-3-mut-BglII, and NF-κB-4-mut-BglII (Table 1 lists all sequences) and their complementary oligonucleotides. To generate the mutation in the AP-1 site when the NF-κB site at −161 already was mutated, the primer NF-κB-2-AP-1-mut-KpnI-EcoRI (Table 1) and its complement were used. The correctness of the introduced mutations in the resulting plasmids pMIEP ΔAP-1, pMIEP ΔElk-1, pMIEP ΔSRF, pMIEP ΔSEE, pMIEP Δ4NF-κB, pMIEP Δ4NF-κB-AP-1, and pMIEP Δ4NF-κB-SEE was confirmed by sequencing.

VR1814 mutants with mutations in the MIEP AP-1, Elk-1, SRF, SEE, and 4NF-κB response elements were generated by a two-step replacement strategy using the galk positive/negative selection method described by Warming et al. (44). Briefly, the FIX-BAC (a gift from T. Shenk) was electroporated into Escherichia coli SW102 (a gift from N. Copeland). In the first step, the galK open reading frame (ORF) was amplified from pgalK (a gift from N. Copeland) by PCR using the MIEP-galk primer set. At the 3′ ends of the forward and reverse primers, specific sequences (of 24 and 20 bp, respectively) dictate the amplification of the galK cassette, and their 5′-end 50-bp tails are homologous to the HCMV VR1814 IE enhancer sequences between nucleotides −52498 and −53104 of the FIX-BAC complete sequence (GenBank accession no. AC146907). Following PCR, the 1,241-bp PCR product was digested with DpnI to remove any plasmid template and gel purified. To accomplish the homologous recombination, approximately 50 ng of DNA was electroporated into SW102 bacteria harboring FIX-BAC. Cells then were plated on minimal medium (M63) agar plates containing 0.2% galactose and chloramphenicol and incubated at 32°C for 5 days. The colonies that appeared were streaked twice on MacConkey agar plates containing 0.2% galactose and chloramphenicol that produced bright red bacterial colonies. One of the single Gal⁺ FIX ΔMIEP-BAC colonies was further characterized for MIEP replacement by PCR and used to initiate the counterselection step. To replace the galK cassette in the second step, the VR1814 MIEP enhancer sequences, each containing one of the different binding site mutations (AP-1, Elk-1, SRF, SEE, or 4NF-κB binding sites), were amplified by PCR from the pMIEP ΔAP-1, pMIEP ΔElk-1, pMIEP ΔSRF, pMIEP ΔSEE, pMIEP Δ4NF-κB, pMIEP Δ4NF-κB-AP-1, and pMIEP Δ4NF-κB-SEE plasmids using the MIEP primer set (Table 1) corresponding to the 3′ ends of the homology arms used in the first selection step. The PCR products were digested with DpnI and gel purified, and 200 ng was electroporated in SW102 cells harboring the FIX ΔMIEP-BAC clone. To select for bacteria with the loss of the galK gene, the transformed bacteria were plated on M63 agar plates containing 0.2% 2-deoxygalactose (DOG) with glycerol as the sole carbon source and chloramphenicol (44). Gal⁻ colonies were characterized for the replacement of galK sequences with the mutated MIEP versions by the PCR amplification of the whole segment, followed by restriction enzyme analysis and sequencing. Two FIX ΔAP-1, ΔElk-1, ΔSRF, ΔSEE, Δ4NF-κB, Δ4NF-κB-AP-1, and Δ4NF-κB-SEE BAC clones were selected and produced. To rescue the mutations of FIX ΔSEE and FIX Δ4NF-κB-SEE, the wild-type (wt) IE enhancer sequence was amplified by PCR from pMIEPGL3 with the same primers set as that used for the amplification of the 4NF-κB- and SEE-mutated sequences. The substitution of the mutated MIEP element in FIX ΔSEE-BAC and in FIX Δ4NF-κB-SEE-BAC then was performed using the two-step replacement strategy and the galK positive/negative selection method as described above. Recombinant FIX ΔSEE REV-BAC and FIX Δ4NF-κB-SEE REV-BAC were characterized for the substitution of the 4NF-κB- and SEE-mutated IE enhancer with the wt MIEP using PCR amplification followed by restriction enzyme analysis and sequencing. Recombinant FIX ΔAP-1, FIX ΔElk-1, ΔSRF, FIX ΔSEE, FIX Δ4NF-κB, FIX Δ4NF-κB-AP-1, FIX Δ4NF-κB-SEE, FIX ΔSEE REV, and FIX Δ4NF-κB-SEE REV viruses, as well as the wt FIX virus, were reconstituted by the electroporation of the corresponding BACs into HELF cells. A plasmid expressing HCMV pp71, the pCMV-pp71 (a gift from T. Shenk), was cotransfected with BAC DNAs.

Nuclear extract isolation and electrophoretic mobility shift assay (EMSA).

HELFs were grown to subconfluence, serum starved, and infected with HCMV AD169 (MOI, 5 PFU/cell). Where indicated, growth-arrested HELFs were treated with U0126 (20 μM; Sigma) for 1 h prior to infection. U0126, a selective inhibitor of MEK1/2 (12), also was present during infection and subsequent incubation periods. At the indicated times p.i., cells were washed in cold PBS, harvested, and centrifuged to collect the pellet. The pellets were incubated for 20 min on ice with a cytoplasmic isolation buffer (10 mM HEPES [pH 7.6], 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.5% NP-40, protease inhibitor cocktail [Sigma]). Samples were centrifuged, and the nuclear pellets were collected by removing the supernatant containing the cytoplasmic extract, washed in cytoplasmic isolation buffer without NP-40, centrifuged, and incubated for 30 min on ice with a nuclear isolation buffer (20 mM Tris-HCl [pH 8.0], 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM PMSF, 25% glycerol, protease inhibitor cocktail [Sigma]). After centrifugation, supernatants containing the nuclear extracts were collected and stored at −70°C.

Electrophoretic mobility shift assays were carried out as previously described (8). Briefly, nuclear extracts (10 μg of proteins) were incubated in a binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM MgCl₂, 5% glycerol) containing 1 μg of poly(dI-dC) (Pharmacia) and the ³²P oligonucleotide probe representing the wild-type HCMV SEE motif (−518 to −547 with respect to the IE1/2 transcription start site). Complexes were analyzed by nondenaturating 4% polyacrylamide gel electrophoresis, dried, and detected by autoradiography. For supershift experiments, 1 μg of rabbit polyclonal antibodies recognizing SRF (sc-335X; Santa Cruz Biotechnology) or 1 μg of a mouse monoclonal against p-Elk-1 (Ser383; sc-8406X; Santa Cruz Biotechnology) was added to the solutions after protein-DNA incubation for 30 min at 20°C, and the DNA-protein complexes were resolved in nondenaturing 4% polyacrylamide gel. Unlabeled 30-bp annealed SEE oligonucleotide was added as the competitor DNA in 300-fold molar excess above the level of the SEE probe.

Immunoblotting.

For immunoblotting, fibroblasts were grown to subconfluence, serum starved, and infected with HCMV AD169 (MOI, 5 PFU/cell). Where indicated, they were treated with U0126 (20 μM; Sigma) for 1 h prior to infection. U0126 also was present during infection and subsequent incubation periods. At the indicated times p.i., whole-cell protein extracts were prepared by resuspending pelleted cells in lysis buffer containing 50 mM Tris-Cl (pH 6.8), 1% sodium dodecyl sulfate (SDS), 1× protease inhibitor cocktail (P8340; Sigma), 1× phosphatase inhibitor cocktail 1 (P2850; Sigma), 1× phosphatase inhibitor cocktail 2 (P5726; Sigma). After being boiled at 95°C for 5 min, soluble proteins were collected by centrifugation at 15,000 × g for 10 min. Supernatants were analyzed for protein concentration with a Bio-Rad Dc protein assay kit (Bio-Rad Laboratories) and stored at −80°C. Proteins were separated by SDS-12% polyacrylamide gel electrophoresis (50 μg of protein per lane) and then transferred to Immobilon-P membranes (Millipore). The filters were blocked in a solution of 5% nonfat dry milk, 10 mM Tris-Cl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 and then immunostained with rabbit polyclonal antibodies against phosphorylated MEK1/2 (p-MEK1/2) (Ser217/221; 9121; Cell Signaling Technology) (diluted 1:500), p-ERK1/2 (Thr202/Tyr204; 9101; Cell Signaling Technology) (diluted 1:1,000), ERK1/2 (9102; Cell Signaling Technology) (diluted 1:1,000), Elk-1 (9182; Cell Signaling Technology) (diluted 1:1,000), SRF (sc-335X; Santa Cruz Biotechnology) (diluted 1:500), or mouse monoclonal antibodies against p-Elk-1 (Ser383; sc-8406X; Santa Cruz Biotechnology), IEA (IE1 plus IE2) (11003; Argene) (diluted 1:250), and UL44 (clone 1202; Goodwin Institute, Plantation, FL) (diluted 1:1,000); mouse monoclonal antibody (MAb) against actin (MAB1501R; Chemicon, Temecula, CA) (diluted 1:2,000) was used as the control for protein loading. Immunocomplexes were detected with sheep anti-mouse or donkey anti-rabbit immunoglobulin antibodies conjugated to horseradish peroxidase (Amersham) and visualized by enhanced chemiluminescence (Super Signal; Pierce).

Inhibition of Elk-1 and SRF protein expression.

HELFs cells were transiently transfected using TransIT-LT1 transfection reagent (OriGene Technologies, Inc.) with 5 μg of either a pRS short hairpin RNA (shRNA) expression plasmid containing a 29-nucleotide sequence insert specific for either Elk-1 (shElk-1 A or shElk-1 B) or SRF (shSRF C or shSRF D) or a pRS plasmid containing a noneffective shRNA cassette against green fluorescent protein (GFP) as a negative control for specific gene downregulation (NCS) (Origene Technologies, Inc.) (Table 1 lists the sequences) and then incubated in high- or low-serum medium for 72 h. Thereafter, total cell extracts were prepared and analyzed by immunoblotting with rabbit anti-Elk-1 or anti-SRF antibodies. To investigate the effects of shRNAs on HCMV IE gene expression, transiently transfected cells were infected with HCMV AD169 (MOI of 3 PFU/cell), and at 24 h p.i. total RNA was isolated and reverse transcribed; real-time reverse transcription-PCR (RT-PCR) was carried out for IE1, IE2, and β-actin (see below).

RT-PCR analysis.

Real-time RT-PCR analysis was performed on an Mx 3000 P (Stratagene) using SYBR green as a nonspecific PCR product fluorescent label. After HCMV infection and cell treatment, total cellular RNA was isolated using Eurozol reagent. RNA (1 μg) then was retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl₂) containing 5 mM random primers, 0.5 mM deoxynucleoside triphosphate (dNTP), and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. cDNAs (2 μl) (or water, as a control) were amplified in duplicate by real-time RT-PCR using the Brilliant SYBR Green QPCR Master Mix (Stratagene) in a final volume of 25 μl. Primer sequences for assessing IE1, IE2, and β-actin mRNA levels are listed in Table 1. Following an initial denaturing step at 95°C for 2 min to activate 0.75 U of Platinum TaqDNA polymerase (Invitrogen), the cDNAs were amplified for 30 cycles (95°C for 1 min, 58°C for 1 min, and 72°C for 1 min). For quantitative analysis, semilogarithmic plots were constructed of the change in fluorescence versus the cycle number, and a threshold was set for the changes in fluorescence at a point in the linear PCR amplification phase (C_T). The C_T values for each gene were normalized to the C_T values for β-actin using the ΔC_T equation. The level of target RNA, normalized to the endogenous β-actin reference and relative to results for the cells infected for 12 h, was calculated by the comparative C_T method and the 2^−ΔΔCt equation.

HCMV infection stimulates TCF formation in quiescent cells and Elk-1 activation through the MEK1/2-ERK1/2 pathway.

Elk-1 can be activated through multiple pathways, including the protein kinase C (PKC), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and the p38/MAPK pathways (14, 26, 31, 35, 46). Although HCMV infection has been reported to stimulate the activity of both the MAPK/ERK1/2 and p38/MAPK pathways (10, 20, 21), it is unclear whether Elk-1 becomes activated. In addition, it is not known whether this activation results in the ability of Elk-1 to stimulate the formation of transcriptionally competent TCF on the SEE of the MIEP in the context of HCMV infection. To address these issues, EMSA analyses were performed using a probe containing the SEE nucleotide sequence (from −518 to −547 relative to IE1/2 transcription start site) and a nuclear extract prepared from quiescent HELFs that had been infected with HCMV for the indicated times (p.i.). As shown in Fig. 1A (left), HCMV infection stimulated a rapid and sustained binding of a protein complex to the SEE probe. Since the transcriptional activation of TCF is known to require the interaction of phosphorylated Elk-1 with both SRF and its cognate SRE (6, 36), supershift analyses using anti-SRF and anti-phospho-Elk-1 antibodies were performed to investigate whether the protein complex formed on the SEE probe was indeed composed of these two transcription factors. The results confirm that the HCMV-induced complex contains both SRF and phosphorylated Elk-1 (Fig. 1A, right). Moreover, the mutation of the SRF binding site within the SEE probe abolished TCF formation in extracts from quiescent HELFs infected with HCMV (data not shown), thus suggesting a role of SRF as the constitutive scaffold protein required to initiate TCF assembly by binding to the SEE and recruiting phosphorylated Elk-1.

To investigate further the mechanisms of Elk-1 activation following HCMV infection, immunoblot analyses were performed on extracts prepared from quiescent cells infected with HCMV at different times p.i. As seen in Fig. 1B, the HCMV infection of quiescent cells stimulated the phosphorylation of Elk-1 within 15 min, and prolonged levels of phospho-Elk-1 were detected up to 24 h p.i. The virus also induced the activation of the upstream ERK1/2 and MEK1/2 kinases with the same two-phases kinetics as those previously described (21), confirming this MAPK cascade to be a virally regulated transduction pathway leading to Elk-1 activation.

Taken together, these results demonstrate that HCMV infection in growth-arrested cells induces a rapid and sustained formation of genuine TCF containing the phosphorylated form of Elk-1 as well as the constitutive SRF protein, and that Elk-1 is regulated by the virus-activated MEK1/2-ERK1/2 pathway.

HCMV-activated MEK1/2 signaling is required for efficient TCF formation and IE and E gene expression in quiescent cells.

The observation that the MEK1/2-ERK1/2 pathway was activated by virus infection within the first hour p.i. suggests that it plays a role in TCF activation and therefore in MIEP activation and the initiation of viral gene expression in quiescent cells. To examine the importance of this HCMV-activated signaling for efficient MIEP activation in quiescent cells, EMSA analyses were performed with the SEE probe and the nuclear extracts prepared from quiescent HELFs infected in the presence of UO126, a potent and specific inhibitor of both MEK1 and MEK2 kinase activity (12). Immunoblot analyses demonstrated that treatment with UO126 (20 μM) for 1 h prior to infection was able to abolish HCMV-induced MEK1/2, ERK1/2, and Elk-1 phosphorylation throughout infection (Fig. 2A). Moreover, as can be seen in the EMSA autoradiograph reported in Fig. 2B, UO126 significantly reduced the formation of virus-mediated TCF, indicating that the HCMV-mediated activation of MEK1/2 is required for optimal TCF assembly above the SEE.

Having established that the inhibition of MEK1/2 by UO126 inhibited both Elk-1 phosphorylation and HCMV-mediated TCF induction, we next examined the consequence of this inhibition upon the IE and E program of HCMV gene expression. In order to do so, the extracts of quiescent HELFs that had been treated with UO126 and infected with HCMV were analyzed for their content of IE and E proteins by immunoblotting with specific antibodies. The expression of IEA (IE1 and IE2) and UL44 was assessed as a control for IE and E proteins. As shown in Fig. 2A, UO126 inhibited the expression of these HCMV proteins. Taken together, these results demonstrate the requirement of virus-activated MEK1/2 signaling for the assembly and activation of the TCF complex and, in turn, for optimal HCMV IE and E gene expression.

Elk-1 and SRF both are required for optimal MIEP activity and IE gene expression in quiescent cells.

Since Elk-1 and SRF constitute the TCF protein partners required for both DNA binding and transcriptional activation, we next investigated the role that these two proteins play in the regulation of MIEP activity in quiescent and proliferating cells. pRS plasmids containing specific 29mer short hairpin RNAs (shRNAs) directed against either Elk-1 or SRF were transfected into HELFs that subsequently were incubated in high- or low-serum medium for 72 h. Total protein extracts then were prepared, and the Elk-1 and SRF protein contents were measured by immunoblotting. As shown in Fig. 3A, the two distinct shRNA sequences directed against Elk-1 (shElk-1 A and shElk-1 B) significantly reduced Elk-1 expression in both quiescent and growing cells without affecting the SRF protein levels. To determine the effects of Elk-1 silencing on the expression of HCMV IE genes, IE1 and IE2 mRNA levels were measured in quiescent and in proliferating HELFs that had been transfected with Elk-1 shRNAs for 72 h and then further infected with HCMV for a further 24 h. As shown in Fig. 3B, both IE1 and IE2 expression levels were significantly reduced in quiescent HELF cells. Since this reduction was not observed in Elk-1-silenced proliferating HELFs, it is clear that Elk-1 activity plays a role in growth-arrested cells for optimal IE gene expression.

When SRF protein expression was reduced by transfecting the shRNA sequence shSRF D (Fig. 4A), a significant decrease in IE1 and IE2 mRNA expression was seen once again in the quiescent but not in the proliferating cells (Fig. 4B). This result again suggests that SRF plays a positive role in the optimization of MIEP activity in growth-arrested cells. This reduction in IE mRNA levels was not observed in HELFs transfected with the shSRF C sequence that was ineffective in reducing SRF protein expression (Fig. 4A), thus supporting the specificity of silencing by shSRF D.

Taken together, these results indicate that the expression of both Elk-1 and SRF is required for optimal MIEP activity and IE gene expression in quiescent cells.

Mutagenesis of SRF and Elk-1 binding sites in the MIEP impairs HCMV replication in quiescent cells.

To investigate the contribution of the TCF complex partners (Elk-1, SRF, and cognate SEE) to HCMV replication in quiescent and proliferating cells, HMCV mutants were produced that contained point mutations in either the Elk-1 or the SRF binding site or in both sites to generate a total inactivation of the SEE (Fig. 5A). Mutations also were introduced into the unique AP-1 site (at −168 relative to the +1 transcription start site) to eliminate its binding to a distinct MIEP element that interacts with growth factor-activated transcription factors (28, 34). To generate these mutants, the MIEP region from HCMV VR1814 DNA was amplified and subjected to site-directed mutagenesis by introducing the restriction site EcoRI into the AP-1 site, EcoRI into the Elk-1 site, KpnI into the SRF site, and EcoRI and KpnI into the SEE (Fig. 5A). These mutations abolished the ability of the transcription factors to bind to their respective sites within the SEE (data not shown). MIEPs, each containing a single mutation (AP-1, Elk-1, SRF, or SEE site), then were used to generate BAC plasmids by using a two-step replacement strategy (44) and the FIX-BAC derived from the HCMV VR1814 strain. Infectious recombinant viruses (RVs) subsequently were reconstituted by the electroporation of the BAC plasmids into HELFs and analyzed for their growth properties.

To determine the viral growth kinetics of the mutant viruses, HELFs in either quiescent or proliferating states were infected with one of the following RVs: RVFIX (parental), RVFIX ΔAP-1, RVFIX ΔElk-1, RVFIX ΔSRF, RVFIX ΔSEE, or the revertant RVFIX ΔSEE REV at an MOI of 0.1 PFU/cell, and titers were determined for infectious virus at 0, 2, 4, 6, 8, 10, and 12 days p.i. As can be seen in Fig. 5B, the replication kinetics of RVFIX ΔSEE in proliferating cells displayed only a minimal defect compared to the kinetics of RVFIX, RVFIX ΔAP-1, and RVFIX ΔSEE REV. In contrast, the RVFIX ΔSEE virus in quiescent cells exhibited a greater-than 2-log decrease in titers compared to those of RVFIX, RVFIX ΔAP-1, and RVFIX ΔSEE REV. Recombinant viruses with either an Elk-1 (RVFIX ΔElk-1) or SRF (RVFIX ΔSRF) mutation exhibited replication defects in quiescent cells similar to those observed with the SEE double mutant (Fig. 5B), indicating the individual contributions of SRF and Elk-1 sites to HCMV replication in growth-arrested cells. Moreover, the reconstitution of the wild-type SEE, as in RVFIX ΔSEE REV, clearly indicates that the replication defect of the ΔSEE virus in quiescent cells was due to the specific disruption of the SRF and Elk-1 sites rather than to alterations in other regions of the HCMV genome.

To gain further insight into the growth defect of the ΔSEE virus in quiescent cells, the expression of IE genes was measured in growing and quiescent cells infected with either RVFIX or RVFIX ΔSEE. As indicated by the real-time RT-PCR analysis shown in Fig. 6, no significant differences in IE1 and IE2 mRNA levels were found in growing cells between the parental RVFIX and the mutant RVFIX ΔSEE at any of the time points analyzed. In contrast, in quiescent cells infected with the ΔSEE virus, IE1 and IE2 mRNA levels were significantly reduced, suggesting that IE gene transcription is impaired in quiescent cells by mutations in the SEE site. Thus, the inefficient replication of the ΔSEE virus in quiescent cells stems from its incapacity to express adequate amounts of pivotal IE proteins. Taken together, these data indicate that the SEE binding element in the MIEP is required for both the transcription of IE genes and productive virus replication in quiescent cells.

SEE-interacting factors compensate for inactivation of the NF-κB sites during viral replication in proliferating cells.

We previously found that the disruption of the four MIEP NF-κB binding sites does not hamper viral replication in growing cells (8). We thus hypothesized that other transcription factors that bind to the MIEP and whose activity is sustained in actively cycling cells could compensate for the inhibition of NF-κB (8). To investigate the potential role of SRF and Elk-1 as compensators, inactivating mutations were introduced into either the SEE or AP-1 site in a MIEP in which the four NF-κB sites also were disrupted (Fig. 7A). To generate these mutants, the MIEP region was amplified from RVFIX Δ4NF-κB DNA (8) and subjected to site-directed mutagenesis by introducing the restriction site EcoRI into the AP-1 site and EcoRI and KpnI into the SEE (Fig. 7A). Recombinant HCMV infectious viruses containing these mutations then were generated and examined for their growth properties. As can be seen in Fig. 7B, the growth of recombinant viruses RVFIX Δ4NF-κB, RVFIX Δ4NF-κB-AP-1, and RVFIX Δ4NF-κB-SEE exhibited, as expected, a severe growth defect when examined in quiescent cells. In contrast, only the RVFIX Δ4NF-κB-SEE virus displayed more than a 2-log decrease in titers in proliferating cells compared to those of RVFIX Δ4NF-κB, RVFIX Δ4NF-κB-AP-1, the revertant RVFIX Δ4ΝF-κΒ-SEE REV, and the parental RVFIX viruses (Fig. 7B).

Finally, to determine whether the growth defect of the Δ4NF-κB-SEE virus in proliferating cells was related to a decrease in MIEP activity, mRNA was extracted from proliferating HELF cells that had been infected with either the RVFIX or RVFIX Δ4NF-κB-SEE viruses and analyzed by real-time RT-PCR for IE1 and IE2 mRNA content. Figure 8 shows that in proliferating cells at 24 h p.i., RVFIX Δ4NF-κB-SEE displayed a 4- and 4.5-fold reduction in IE1 and IE2 mRNA expression levels, respectively, compared to that of parental RVFIX. Taken together, these results indicate that the increased activities of Elk-1 and SRF and their sustained binding to the SEE in cells stimulated by growth factors compensate for the effects of the disruption of the MIEP NF-κB sites.