INTRODUCTION

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Members of the species-specific cytomegalovirus (CMV) family possess potent transcriptional enhancers upstream of their major immediate-early promoters (MIEPs) (6, 10, 40, 41). The MIEP is one of the first promoters to activate upon infection driving expression of key regulatory immediate-early (IE) proteins of the virus. In addition, stringent regulation of its activity in vivo implicates an important role for the MIEP in viral cell-type tropism (4, 5, 22). Thus, transcriptional regulation of the MIEP by the enhancer is believed to play a pivotal role in determining the outcome of a productive infection (13). The MIEP enhancers of the CMV family are highly complex and contain multiple arrays of interdigitating repeat and unique sequence elements (reviewed in reference 13). The arrangement of these regulatory modules and overall sequence composition of the enhancers differ considerably between the different species strains (6, 10, 40). The majority of cellular transcription factors known to interact with the enhancer elements are regulated through signal transduction pathways. In many instances, the same signal-regulated transcription factors have been shown to interact among the different CMV enhancers (e.g., references 2, 3, and 9 and references therein). It therefore appears that while these enhancers differ in primary sequence structure, their functional regulatory elements are highly related. In this connection, it is noteworthy that many of the different species members of the CMV family also share many biological characteristics.

A limited number of mutations have been characterized in regions closely associated with the MIEP enhancer in both murine (MCMV) and human (HCMV) CMVs (7, 29, 30, 32, 34). In HCMV and MCMV, genetic disruption of the ie1 open reading frames (ORFs) (whose transcripts are initiated from their MIEPs) have shown that while the ie1 protein is nonessential for viral growth, it is necessary for optimal infection (15, 32, 34). Importantly, few mutations have been successfully constructed in the MIEP locus, and so far none have been generated in the enhancer region. In HCMV, a region immediately upstream of the enhancer encompassing the modulator region of the MIEP has been deleted and shown not to be essential for MIEP activity in infected differentiated and undifferentiated NT2 cells and human foreskin fibroblasts (30). It therefore appears that mutations in regions closely associated with MIEP enhancer are nonessential for determining a productive infection. However, it is likely that the lack of characterized mutants in the enhancer region occurs because of the difficulty in generating virus mutants with growth disadvantages. Recently, we (M.M. and U.K.) have reported a novel strategy for the genetic manipulation of the MCMV genome, based on the cloning of the infectious viral genome as a bacterial artificial chromosome (BAC) in Escherichia coli (32). This approach provides a useful system for the study of virus mutants with growth defects. To date, the predicted importance of the enhancer of CMV to infection remains untested.

In this study, we show by using the MCMV BAC system, that the enhancer of MCMV plays an important role in promoting a productive infection. We also show that wild-type growth characteristics can be completely restored to enhancerless virus, by linking in cis the homologous MCMV or heterologous HCMV enhancer. Moreover, the genetic complementation studies with the human enhancer provide direct evidence that the enhancer is not involved in restricting the species-specific host range of a CMV infection.

	MATERIALS AND METHODS

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Virus and cells. Murine NIH 3T3 fibroblasts (ATCC CRL1658) were propagated in Dulbecco's modified essential medium supplemented with 2 mM glutamine, 100 U of penicillin per ml, 100 µg of gentamicin per ml, and 10% calf serum. Mouse embryonic fibroblasts (MEFs), derived from the embryos of timed pregnant BALB/c.ByJ mice on day 19 of gestation, were cultured in the same medium as NIH 3T3 cells, except that 10% fetal bovine serum was used instead of calf serum. The Smith strain of MCMV, originally obtained from Ann Campbell (Eastern Virginia Medical School), was used as the parental wild-type MCMV in this study. Stocks of MCMV were prepared in NIH 3T3 cells, and titers were determined by standard plaque assay on NIH 3T3 cells.

Plasmid constructs. Recombinant plasmids were constructed according to established procedures (28). Plasmid pBam25 containing the sequences from 176441 to 187035 of the MCMV genome placed into the BamHI site of pACYC184 has been described previously (named pAMB25 in reference 18). The construction of pBam25H was done as follows: primers dNN and BlpI were used to amplify a 616-bp fragment from the HCMV enhancer with pMIEP(1114/+112)CAT as a template (14). Primer dNN (5'-gcc ctt taa atg cat aaa gcg gcc gct gca tac gtt gta tcc ata tc-3') contains a NotI site, an NsiI site, and a DraI site adjacent to nucleotide 667 (relative to the ie1/ie2 HCMV transcription start site) of the HCMV enhancer. Primer BlpI (5'-gta cgc tca gcc cgc cca ttt gcg tca atg ggg c-3') contains an EspI site adjacent to nucleotide 52 (relative to the ie1/ie2 HCMV transcription start site) of the HCMV enhancer. The resulting PCR fragment was digested with EspI and DraI and inserted into NdeI-EspI-digested pON401 (with the NdeI site filled in [29]) to generate pON401H. A 2.7-kbp MluI fragment of pON401H was inserted into MluI-digested pBam25 to create pBam25H.

BAC mutagenesis. Mutagenesis of the MCMV BAC plasmid pSM3 was performed as described previously (32). In brief, shuttle plasmids were electroporated into E. coli CBTS bacteria (20) that already contained the BAC plasmid pSM3. Note that E. coli CBTS is RecA⁺ at 30°C and virtually RecA at temperatures higher than 37°C (the CBTS strain was constructed by M. O'Connor, University of California Irvine). Transformants were selected at 30°C on Luria broth (LB) plates containing chloramphenicol (12.5 µg/ml) and tetracycline (10 µg/ml) or chloramphenicol with ampicillin (50µg/ml), using shuttle plasmid pMB097 or pST76-A, respectively. Clones that formed cointegrates were identified by streaking the bacteria on new LB plates followed by incubation at 43°C. To allow resolution of the cointegrates, clones were streaked on LB plates containing chloramphenicol only and incubated at 30°C. Bacteria were restreaked on LB plates with chloramphenicol to separate clones that contain resolved plasmids and clones that still contain cointegrates. Clones with resolved plasmids were identified by screening for the loss of the antibiotic marker of the shuttle plasmid. Usually, about 5 to 10% of the clones resolved the cointegrates. Finally, BAC plasmid DNA was isolated from 10-ml overnight cultures by the alkaline lysis procedure (28) and characterized by restriction enzyme digestion to identify BAC plasmids that received the mutation. Midi preparations of BAC plasmids were prepared from 100-ml E. coli cultures as described previously (28, 32).

Transfections and virus construction. To generate recombinant viruses, BAC plasmids were transfected, in the absence or presence of pBam25 or pBam25H, into NIH 3T3 cells (in six-well dishes) by the calcium phosphate precipitation technique essentially as described previously (28). Six hours posttransfection, cells were treated with glycerol (15% glycerol in HEPES-buffered saline) for 3 min as described previously (28). The progeny virus that replicates in these cultures was harvested when 100% cytopathic effect was observed in the cultures and then was used to infect fresh cells. Two independent recombinants of the hMCMV-ES (from two independent cotransfections of pE1 with pBam25H) hybrid and one recombinant MCMVrev (from a cotransfection of pE1 with pBam25) were subjected to three rounds of plaque purification before a high-titer stock was prepared.

Viral infections. NIH 3T3 cells or MEFs were infected with wild-type MCMV or different MCMV recombinants at the different multiplicities of infection (MOIs) indicated in the figure legends. After a 1-h adsorption period, the virus inoculum was removed, the cultures were washed three times with phosphate-buffered saline, and fresh medium was added. At different times after infection, the supernatants of three independent cultures were harvested, frozen, and thawed, and infectious virus was quantitated by standard plaque assay on NIH 3T3 cells. For selective expression of IE transcripts, the cultures were incubated from 30 min prior to infection and up to 12 h postinfection in the presence of cycloheximide (100 µg/ml).

DNA and RNA analysis. Total cell DNA was isolated from infected NIH 3T3 cells (in 60-mm-diameter tissue culture dish) when 100% cytopathic effect was observed in the cultures. Briefly, infected cells were scraped, washed twice with phosphate-buffered saline, and lysed in 1 ml of a solution containing 10 mM Tris (pH 7.8), 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 250 µg of proteinase K per ml, and incubated at 55°C for 4 h. DNA was then purified by phenol-chloroform extraction and precipitated with ethanol. When DNA restriction enzyme patterns were analyzed, viral DNA fragments after restriction enzyme digestion were run on 0.5% agarose gels for approximately 18 h at 0.5 V/cm. DNA blotting was conducted as described previously (28). The HCMV enhancer-specific probe used was a 0.34-kbp SpeI-SnaBI fragment from pMIEP(1145/+112)CAT. Whole-cell RNA was isolated from either uninfected or infected NIH 3T3 cells (in six-well dishes) by using RNAzol B (Tel-Test, Inc., Friendswood, Tex.) according to the manufacturer's protocol. Northern blot analyses were performed as described in reference 28. Two to eight micrograms of RNA was separated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde, transferred to a nylon membrane, and hybridized with a 1.6-kbp MluI-HindIII fragment from pON401 to specifically detect ie1/ie3 transcripts (29). The probes used were isolated from agarose gels and then radiolabeled with [-³²P]dATP by the random-primed labeling method (12).

Immunoblot analyses. NIH 3T3 cells (in six-well dishes) were infected with MCMV at an MOI of 0.5 PFU/cell. At different times after infection, samples were lysed in protein sample buffer, vortexed, and boiled for 5 min. The polypeptides of cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8% polyacrylamide) and transferred to nitrocellulose filters. Filters were incubated with an ie1-specific monoclonal antibody, Croma 101, and as a secondary antibody horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Amersham, Buckinghamshire, England) was used. Blots were developed with the Enhancer chemiluminescence system (Amersham) according to the manufacturer's protocol.

	RESULTS

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Generation of enhancerless MCMV genomes. To determine if the enhancer is essential for efficient viral growth in tissue culture cells, a series of recombinant MCMV genomes lacking enhancer sequences were constructed by using the recently described MCMV BAC system (32). In this system, the MCMV genome has been cloned as a BAC in E. coli, and viral progeny can be reconstituted by transfecting the MCMV BAC plasmid into eukaryotic cells permissive for MCMV. As a parental BAC plasmid for the generation of BACs carrying enhancerless viral genomes, we used pSM3. The BAC plasmid pSM3 contains the MCMV genome with BAC vector sequences replacing a nonessential region for replication in vitro within the HindIII E' fragment at the right-terminal end of the viral genome (spanning nucleotides 209756 to 217934). A schematic representation of the various BAC recombinant genomes constructed is summarized in Fig. 1. In the first MCMV BAC plasmid, pE1, an MunI-HpaI fragment of approximately 1.6 kbp in the HindIII L fragment of parental BAC pSM3 is deleted. Deletion of this 1.6-kbp fragment, which encompasses nucleotide sequences from +209 to 1421 of the IE region, effectively removes the complete MIEP regulatory region and also disrupts the first exon of ie1/ie3. To generate a less extensive disruption of the MIEP in which only enhancer sequences have been removed, a 1.1-kbp EspI-NdeI sequence (within the HindIII L fragment) extending from nucleotide positions 48 to 1191 of the MCMV MIEP was resected from pSM3 to construct the enhancerless BAC plasmid pSM3dE (see Fig. 1 and Materials and Methods for details). In addition, and to maintain the relative positions of the promoters flanking the MCMV enhancer, a BAC plasmid, pSM3dE::luc, that contains a stuffer fragment replacing the deleted enhancer segment was generated (Fig. 1). In pSM3dE::luc, an internal nonregulatory 770-bp fragment from the firefly luciferase reporter gene was inserted within nucleotides 48 and 1191 of the enhancer region of MCMV. To verify the structure of the recombinant BAC plasmids generated, their HindIII restriction patterns were analyzed. As shown in Fig. 2, in comparison with the parental BAC plasmid pSM3, the recombinant enhancerless MCMV BAC plasmids lack the wild-type 7.1-kbp HindIII L fragment, which instead is replaced by the expected 5.5-kbp fragment in pE1, 6.0-kbp fragment in pSM3dE, and 6.7-kbp fragment in pSM3dE:luc. In addition, comparison of the XbaI and EcoRI fragment profiles with those of parental BAC pSM3 indicated that pE1, pSM3dE, and pSM3dE::luc were free of any detectable deletions or insertions in other regions outside the HindIII L fragment of the viral genome (data not shown). These results demonstrate the successful deletion of the enhancer and its replacement by a stuffer fragment in the recombinant BAC plasmids pE1, pSM3dE, and pSM3dE::luc, respectively.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Construction of enhancerless MCMV BAC genomes. The top line represents the HindIII map of the wild-type (Wt) MCMV genome, with the HindIII K and L regions expanded below to show the region containing the MIE genes (ie1 to ie3). The MCMV BAC plasmids pE1, pSM3dE, and pSM3dE::luc, shown below the wild-type MCMV genome, were generated by homologous recombination in E. coli with pSM3 as the parental MCMV BAC genome as indicated in Materials and Methods. A MunI-HpaI fragment (from +209 to 1421 relative to the ie1/ie3 MCMV transcription start site) in the HindIII L fragment of the wild-type MCMV genome was removed to generate pE1. In pSM3dE, an EspI-NdeI fragment (from nucleotide sequences 48 to 1191 relative to the ie1/ie3 MCMV transcription start site) was deleted in the HindIII L fragment of the wild-type MCMV genome. The MCMV BAC plasmid pSM3dE::luc contains a 770-bp fragment from the luciferase gene replacing nucleotide sequences from 48 to 1191 (relative to the ie1/ie3 MCMV transcription start site) in the HindIII L fragment of the wild-type MCMV genome. The illustration is not drawn to scale.

View larger version (66K): [in this window] [in a new window]   FIG. 2.   Structural analysis of enhancerless MCMV BAC genomes. Ethidium bromide-stained agarose gels of HindIII-digested BAC plasmids pSM3 (1), pE1 (2), pSM3dE (3), and pSM3dE::luc (4) after separation on a 0.5% agarose gel. The HindIII fragment name (11) is indicated to the right of the set of lanes, and the size markers are shown at the left margin. The size of the new HindIII L fragments is indicated with an arrow at the right margin.

Requirement of the enhancer region for viral DNA infectivity. To test directly the significance of the enhancer to infection, recombinant MCMV BAC genomes either containing (pSM3) or lacking the MIEP enhancer region (pE1 and pSM3dE) were prepared from bacteria and transfected into NIH 3T3 cells, and viral DNA infectivity was assessed by plaque formation. Note that in pE1, the entire MIEP region has been deleted, whereas in pSM3dE, the MIEP is truncated at position 48 and therefore lacks exclusively enhancer sequences while retaining the wild-type MIEP core promoter (TATA box) element. The results from this set of experiments are shown in Table 1. As expected, numerous plaques developed in NIH 3T3 cells when transfected with the wild-type MCMV BAC plasmid, pSM3, and cultures reached a complete cytopathic effect within 9 days. In contrast, NIH 3T3 cells transfected with the recombinant MCMV genome containing the large 1.6-kbp deletion in the MIEP enhancer, pE1, failed to develop any plaques. These results suggest that elimination of the complete MIEP, including part of exon 1 of ie1/ie3, generates a replication-incompetent virus. When NIH 3T3 cells were transfected with pSM3dE, a minimal number of small plaques were produced, and cultures took more than twice as long (24 days) to reach cytopathic effect (Table 1). Similar results were achieved by transfecting the MCMV BAC plasmids into MEFs (data not shown). Altogether, the results of these experiments are consistent with the enhancer being important for optimal infectivity.

Complementation of viral DNA infectivity of enhancerless MCMV by transfection of cells with an ie1/ie3 expression plasmid. In the next set of experiments, we sought to determine whether the defect in infectivity observed in the enhancer-deficient genomes is due to genetic alteration of the IE locus alone. For this purpose, we used a recombinant plasmid, pBam25, that expresses ie1/ie3 genes upon transfection in NIH 3T3 cells (Fig. 3 and data not shown). In the presence of pBam25, approximately equal numbers of plaques were developed for each enhancerless recombinant compared with reconstituted MCMV derived from pSM3 (Table 1). These results demonstrate that the failure of enhancerless recombinants to efficiently form plaques is due to a specific alteration of the IE locus.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Construction of hMCMV-ES mutants. The top line represents the HindIII map of wild-type MCMV genome, with the HindIII K and L regions expanded below to show the region containing the MIE genes (ie1 to ie3). In the MCMV BAC plasmid pE1, a MunI-HpaI fragment (from nucleotide sequences +209 to 1421 relative to the ie1/ie3 MCMV transcription start site) has been deleted in the HindIII L region of the wild-type MCMV genome. Recombinant viruses MCMVrev and hMCMV-ES were constructed by cotransfection of MCMV BAC plasmid pE1 with pBam25 or pBam25H, respectively, into NIH 3T3 cells. pBam25, a plasmid expressing ie1 and ie3, carries a BamHI fragment containing sequences from 176441 to 187035 of the MCMV genome. pBam25H carries the BamHI fragment (containing sequences from 176441 to 187035 of the MCMV genome) in which sequences from 48 to 1191 (relative to the ie1/ie3 MCMV transcription start site) have been replaced by sequences from 52 to 667 of the HCMV enhancer. The solid and hatched boxes represent the MCMV and HCMV enhancers, respectively. The diagram is not drawn to scale.

View larger version (66K): [in this window] [in a new window]   FIG. 4.   Structural analysis of hMCMV mutants. DNA isolated from NIH 3T3 cells infected with wild-type MCMV (1), MCMVrev (2), hMCMV-ES1 (3), or hMCMV-ES2 (4) was subjected to HindIII digestion and separated on a 0.5% agarose gel. Bands were visualized with ethidium bromide (A) or transferred to nylon filters and hybridized to a 32P-labeled 340-bp SpeI-SnaBI fragment from the HCMV enhancer (B). The HindIII fragment name (11) is indicated to the left set of lines, and the sizes of the natural and new HindIII L fragments for each virus are shown with an arrow.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Growth kinetics of hMCMV-ES mutants. Murine NIH 3T3 cells were infected at an MOI of 5 (A) or 0.01 PFU (B) per cell with wild-type (Wt) MCMV (Smith strain), MCMVrev, hMCMV-ES1, or hMCMV-ES2. At the indicated time points (hours) after infection (hpi) supernatants from the infected cultures were harvested, and titers were determined by plaque assay on NIH 3T3 cells. Each data point represents the average and standard deviation from three separate cultures.

Genetic complementation and restoration of growth of enhancerless MCMV by the HCMV enhancer. Since the enhancer region of MCMV contains a number of putative ORFs (m124, m124.1, and m125) (38), it is possible that the loss of these MCMV-specific sequences may be responsible, in part, for the severely impaired ability of enhancerless MCMV to grow. In order to test this hypothesis and to explore the possibility of whether the human enhancer can rescue growth, we sought to construct a hybrid virus that substituted the MCMV enhancer for the HCMV enhancer. For these experiments, we first constructed a plasmid, pBam25H, containing the MCMV-MIE region in which sequences from 48 to 1191 of the MCMV enhancer were replaced by the paralogous sequences from 52 to 667 of the HCMV enhancer (Fig. 3 [see Materials and Methods for details]). We next tested the ability of pBam25H to complement in trans the growth defect of the enhancerless BAC plasmid (pE1) in transient cotransfection assays. In these experiments, cotransfection of pE1 and pBam25H in NIH 3T3 cells led to the development of plaques that took around 9 days to reach a complete cytopathic effect. Therefore, these results indicate that the severe deficiency of enhancerless MCMV to grow is not due to the loss of expression from one of the putative ORFs (m124-m125) that overlap the enhancer region and further suggest that the HCMV enhancer may complement an enhancerless MCMV.

View larger version (67K): [in this window] [in a new window]   FIG. 6.   Expression of RNA kinetics of ie1/ie3 transcripts by hMCMV-ES mutants. NIH 3T3 cells were mock infected (lane 1) or infected at an MOI of 0.5 PFU/cell with wild-type MCMV (lanes 2, 6, 10, and 14), hMCMV-ES1 (lanes 3, 7, 11, and 15), hMCMV-ES2 (lanes 4, 8, 12, and 16), and MCMVrev (lanes 5, 9, 13, and 17). Whole-cell RNA was harvested at the indicated times after infection in the presence (lanes 2 to 5) or absence (lanes 1 and 6 to 17) of CH, separated on a formaldehyde-agarose gel, transferred to a nylon membrane, and probed with a 1.6-kb MluI-HindIII fragment from pON401 that was specific for ie1 transcripts. The position of the 2.75-kb transcripts is indicated on the right by an arrow. The positions of the 18S and 28S rRNAs are shown. The blot was then hybridized with a 32P-labeled GAPDH probe as an internal RNA control. The 1.4-kb GAPDH band detectable in all lanes is shown on the bottom panels.

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Expression kinetics of ie1 protein in hMCMV-ES-infected cells. NIH 3T3 cells were mock infected (lane 1) or infected at an MOI of 0.5 PFU/cell with wild-type MCMV (lanes 2, 6, 10, and 14), hMCMV-ES1 (lanes 3, 7, 11, and 15), hMCMV-ES2 (lanes 4, 8, 12, and 16), and MCMVrev (lanes 4, 9, 13, and 17). At the indicated times after adsorption, samples were lysed, subjected to SDS-PAGE analysis on 8% polyacrylamide gels, and reacted with an ie1 monoclonal antibody. Molecular mass standards (M) appear on the left. The position of the 89-kDa protein is indicated by an arrowhead.

	DISCUSSION

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This study shows that the enhancer region of MCMV plays a critical role for efficiently effecting productive infection in tissue culture cells. We further demonstrate that genetically exchanging the MCMV enhancer with that from HCMV effectively produces wild-type growth characteristics. These results have a number of important implications for understanding and exploiting the role of the enhancer in the CMV infectious program.

Enhancer is important for MCMV growth. The results of this study clearly show that enhancer-deficient strains or recombinant genomes of MCMV are severely compromised in their ability to synthesize virus in infected or transfected cells. Previous studies have indicated that enhancers play a number of distinct roles in a viral life cycle. The most important role served by a viral enhancer is in the initiation of the infectious program by which it acts as a potent transcriptional activator region for the viral IE genes. In agreement, our experiments showing that a related but distinct enhancer can efficiently substitute for the naturally occurring enhancer strongly suggest that a primary function of this region is to increase the efficiency of IE gene expression by serving as a potent activator of transcription. These enhancer swap experiments might also underscore the functional importance of the conserved regulatory elements within the HCMV and MCMV enhancers, although we predict that any enhancer could substitute, in part, for the CMV enhancer. Viral enhancers have also been implicated in the maintenance of an open chromatin structure prior to and after the initiation of replication (8). In this connection, we note that in an infection of permissive cells by HCMV, marked DNase I hypersensitivity is observed in the enhancer region, indicating an open chromatin structure at early times of infection (35). In addition, viral enhancers may directly potentiate viral DNA replication (16, 17, 36) or may even be involved in localizing virion DNA to preferred nuclear compartments. It is conceivable that all of these possibilities may be responsible for the requirement of the enhancer for optimal MCMV infection. Whether the enhancer plays any direct role in promoting MCMV DNA replication or modifying chromatin structure remains to be determined.

Enhancer is not involved in the species restriction of CMV in tissue-culture cells. The CMV family is highly species specific. In this regard, the role of an enhancer in initiating the transcriptional program of a virus can, in principle, limit the virus to productively infect a specific host or cell type (23, 25, 27, 33). Previous in vitro and in vivo studies have indicated that the HCMV enhancer functions appropriately in mouse cells (4, 5, 22, 24), indicating that the strict species specificity of CMV is not at the level of the MIEP. In agreement with this notion, we provide in this study direct evidence that the enhancer is not responsible for the species-specific restriction in the ability of CMV to productively infect cells. At present, we cannot rule out the possibility that the species-specific enhancers may restrict in vivo growth of CMV, although the transgenic studies with the human enhancer indicate that this is unlikely to be the case (4, 5, 22). Indeed, our preliminary experiments indicate that hMCMV strains are capable of productively infecting the mouse (1).

The new hMCMV strains and future implications. In this study, we show that the HCMV enhancer can completely restore the growth deficiency of an enhancerless MCMV. Importantly, these chimeric (hMCMV-ES) strains represent an MCMV that is under the control of HCMV genetic elements and thus may provide the development of a new model for exploring the in vivo importance of select HCMV enhancer elements during acute, latent, and reactivated infections. In particular, mutations introduced in specific binding sites for transcription factors used by the HCMV enhancer will clarify the involvement of the various signal-regulated transcription factors in controlling aspects of viral pathogenesis and latency. Finally, the observation that viral growth can be inhibited by eliminating the enhancer might have important implications in the development of novel ligand-specific drugs for therapy against CMV. For instance, it is possible to generate molecules to recognize specific base pairs in the minor groove by using hairpin polyamides (42) and the major groove by using DNA-binding antibodies (26) or zinc finger chimeras (21).