Subversion of cell cycle regulatory pathways.

Human cytomegalovirus (HCMV) has evolved numerous strategies to commandeer the host cell for producing viral progeny. The virus manipulates host cell cycle pathways from the early stages of infection to stimulate viral DNA replication at the expense of cellular DNA synthesis. At the same time, cell cycle checkpoints are by-passed, preventing apoptosis and allowing sufficient time for the assembly of infectious virus.

The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts.

The isolation of viruses with mutations in essential genes requires that they be propagated in cells expressing the wild-type proteins. This has been a particularly challenging problem for studying mutations in the human cytomegalovirus (HCMV) immediate early (IE) gene, IE2 86. In the past, we tried a number of approaches to derive human fibroblasts expressing wild-type IE2 86, but were unable to maintain expression of a fully functional protein. To overcome this obstacle, we developed a strategy whereby recombinant baculoviruses were used as vectors for the expression of HCMV IE proteins in primary human fibroblasts (FFs). The IE2 86 and IE1 72 cDNAs, as well as the genomic fragment of the UL122-123 region under the control of a chicken actin promoter, were introduced into the baculovirus genome by site-specific transposition in Escherichia coli. Recombinant "bacmid" DNAs were then transfected into Sf9 cells to generate recombinant baculoviruses. FFs infected at high m.o.i. with these baculoviruses expressed high levels of the HCMV protein for at least 1 week, as determined by immunofluorescence assays and Western blots. Moreover, the IE2 86 protein was found to be fully functional with respect to its ability to activate the HCMV UL112-113 early promoter. Recombinant baculoviruses expressing IE1 72 were also able to efficiently complement HCMV ie1 mutants. These data demonstrate the potential of using recombinant baculoviruses as vectors for the expression of toxic viral genes in human cells and for subsequent isolation of mutant HCMV lacking these essential genes.

Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65).

The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.

Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus.

Human cytomegalovirus is a ubiquitous human pathogen that is the leading viral cause of birth defects. It also causes significant morbidity and mortality in both chemically and virally immunosuppressed individuals. Recent studies have begun to elucidate the interplay between this virus and its host cell on a molecular level. The interactions begin upon contact with the cell membrane, involve multiple processes including cell signaling, cell-cycle control and immune response mechanisms, and culminate in a productive infection.

Specific chromosome 1 breaks induced by human cytomegalovirus.

Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem for immunocompromised individuals. Here we show that infection of cells with HCMV during the S-phase of the cell cycle results in two specific chromosome 1 breaks at positions 1q42 and 1q21. We demonstrate that purified virions, and not infected cell supernatant alone, are responsible for the damage. In addition, we show that the specific breaks occur when different sources of fibroblasts and strains of HCMV are used. Incubation of the virus with neutralizing antibody prevents the induction of breaks. However, UV-inactivated virus is as efficient as untreated virus in inducing specific damage to chromosome 1. Thus, there is a requirement for viral adsorption/penetration, but not new viral gene expression. This HCMV-mediated induction of site-specific damage in actively dividing cells may provide clues for the development of neurological defects in the congenitally infected infant.

Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb-related protein p130.

We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G(1), and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729-3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4-DP-1-p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.

In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83).

We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.

Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection.

Expression of many early viral genes during human cytomegalovirus (HCMV) infection is dependent on cellular transcription factors. Several immediate-early and early viral promoters contain DNA binding sites for cellular factors such as CREB, AP-1, serum response factor, and Elk-1, and these transcription factors can be activated by phosphorylation via the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. To determine if the extracellular signal-regulated MAPKs, ERK1 and ERK2, play a role in transcription factor activation during infection, we tested for ERK activity during viral infection. We found that HCMV infection resulted in the maintenance of previously activated ERK1 and ERK2 by a mechanism which appears to involve the inhibition of a cellular phosphatase activity. ERK phosphorylation and activity were sustained for at least 8 h after infection, whereas in mock-infected cells, ERK activity steadily declined by 1 h postinfection. The activity of at least one cellular substrate of the ERKs, the protein kinase RSK1, was also maintained during this period. UV inactivation experiments suggested that viral gene expression was required for sustained ERK activity. In turn, activation of the ERKs appeared to be important for viral gene expression, as evidenced by the observed decrease in the transcriptional activity of the HCMV UL112-113 promoter during infection in the presence of the MEK inhibitor PD98059. These data suggest that HCMV utilizes cellular signal transduction pathways to activate viral or cellular transcription factors involved in the control of early viral gene expression and DNA replication.

Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription.

Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G0, G1, and S). In all cases, infection led to cell cycle arrest. Cells infected in G0 or G1 phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G0, G1, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.

Separate DNA elements containing ATF/CREB and IE86 binding sites differentially regulate the human cytomegalovirus UL112-113 promoter at early and late times in the infection.

The human cytomegalovirus (HCMV) UL112-113 promoter represents a useful model for studying temporal regulation of viral gene expression. Stimulation of this promoter by the 86-kDa immediate-early protein (IE86) is controlled by sequences between nucleotides -113 and -59, which include both an ATF/CREB and an IE86 binding site. In transient assays, the ATF/CREB site is essential, and the IE86 site, although nonessential, can enhance transcription. With recombinant viruses, we have assessed the function of these promoter elements in the context of the viral genome. Transcription from the inserted UL112-113 promoter shows the same temporal pattern as the endogenous promoter, including the switch to an upstream RNA start site late in infection. Deletion of sequences containing the IE86 site results in a decrease in the level of early transcription and elimination of late transcription. In contrast, when the ATF/CREB site is deleted, early RNA synthesis is almost completely abolished, but late transcription is comparable to that of the wild type, with repositioning of the RNA start site downstream by the number of nucleotides deleted. Replacement of sequences between -108 and -95 with the HCMV cis-repression signal from the major immediate-early promoter had no effect on the level of late RNAs but resulted in the repositioning of the RNA start site 39 nucleotides upstream. These results suggest that the ATF/CREB site is functional only at early times, while sequences containing the IE86 site modulate the level of early RNAs and may be required for activating late transcription in a distance-dependent manner.

p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus.

Previously, we reported that human cytomegalovirus (HCMV) infection of fibroblasts markedly affects p53 and other regulatory proteins and inhibits transit through the cell cycle (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). Although the p53 steady-state levels are elevated throughout the infection, evidence suggests that the ability of p53 to transactivate some of its downstream targets is compromised. To elucidate the mechanisms governing the accumulation of p53, we examined the synthesis, stability, and localization of the protein in HCMV-infected fibroblasts. Synthesis of p53 was not increased in the infected cells during the first 24 h postinfection. In fact, pulse-chase experiments revealed that synthesis of p53 in infected fibroblasts was lower than in mock-infected cells. However, after an initial decay, the p53 was stabilized. In addition, beginning at approximately 30 h postinfection, p53 was localized to discrete foci within the nuclei of infected cells. The morphology of these foci suggested that they were replication centers. We confirmed that these are sites of DNA replication by demonstrating both incorporation of bromodeoxyuridine and localization of UL44 (the viral polymerase processivity factor) into these centers. The single-stranded DNA binding protein RPA was also sequestered. In contrast, Rb and HCMV IE1 72 remained distributed throughout the infected cell nuclei, indicating specific targeting of certain proteins. Taken together, our results provide two alternative mechanisms to account for the increased steady-state levels of p53 observed in HCMV-infected fibroblasts.

Molecular analysis of the differential restriction of human immunodeficiency virus type 1 replication in neuronal cell lines.

Human immunodeficiency virus type 1 (HIV-1) replication is restricted partially in SK-N-MC and completely in SK-N-SH neuronal cells. To investigate the molecular mechanism of this differential restriction of HIV-1 replication, cells infected with HIV-1 were analysed for their steady-state levels of: total and unintegrated HIV-1 DNA by DNA PCR, different species of HIV-1 RNA by RT-PCR, and HIV-1 p24 protein production by an ELISA procedure. We found that the kinetics of the infection were slower and there was a lower level of accumulation of HIV-1 macromolecules (total and unintegrated circular DNA, unspliced and spliced RNAs and viral proteins) in the SK-N-MC cells than in the permissive CEM cells. In SK-N-SH cells, HIV-1 DNA was only transiently detected during the first 24 h post-infection, and the unspliced RNA was detected up to 1 week post-infection. However, the HIV-1 spliced RNAs and the 2-LTR circular DNA were not detected at all during the course of infection. Both SK-N-MC and SK-N-SH cells showed higher levels of HIV-1 DNA, RNA and p24 protein when infected with an HIV-1 (amphotropic retrovirus) pseudotype, HIV-1B. However, the level of HIV-1 replication was still lower in SK-N-SH than in SK-N-MC cells. Moreover, although the kinetics of viral protein production were comparable in SK-N-MC cells infected with HIV-1B and CEM cells infected with HIV-1, the overall level of virus replication was still much lower in HIV-1B-infected SK-N-MC cells. These data suggest that the restriction of HIV-1 replication in neuronal cell lines takes place at both virus-entry and post-entry levels, and cellular factors may be involved in the differential restriction of HIV-1 replication in these cells.

Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other.

The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis reveals that there are two domains on IE2 86 that can independently affect Rb function. One region (aa 241 to 369) includes the major Rb-binding domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact with each other via at least two separate domains and provide further support for the hypothesis that IE2 86 may exert its pleiotropic effects through the formation of multimeric protein complexes.

A model system for human cytomegalovirus-mediated modulation of human immunodeficiency virus type 1 long terminal repeat activity in brain cells.

Previously, our laboratory showed that human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) in brain-derived cells with limited HIV-1 gene expression but inhibits HIV-1 in cells fully permissive for replication of both viruses (F. M. Jault, S. A. Spector, and D. H. Spector, J. Virol. 68:959-973, 1994). To investigate these effects further, we developed a model system that uncouples HIV-1 gene expression from long terminal repeat (LTR) activity. Two monoclonal U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) were generated, each containing an integrated copy of an LTR-chloramphenicol acetyltransferase (CAT) construct and the Escherichia coli lacI gene. LIGHIVDC also has an inducible HIV-1 genome controlled by a Rous sarcoma virus promoter with lac operator sequences. Basal LTR-mediated CAT activity is 65-fold higher in LIGHIVDC than in LTRIG, and this activity is further increased (20-fold) following incubation of LIGHIVDC with isopropyl-beta-D-thiogalactopyranoside (IPTG). Tat protein can be detected by immunostaining in LIGHIVDC. However, Rev-mediated transport and subsequent translation of the singly spliced and unspliced HIV-1 mRNAs is inefficient. In the absence of Tat, HCMV stimulated CAT activity approximately 20-fold, and this activation required HCMV gene expression but not viral DNA replication. LTR-directed transcription was unaffected by HCMV infection in LIGHIVDC but was inhibited in these cells when they contained increased Tat levels following IPTG induction. These results support the hypothesis that HCMV can induce the HIV-1 LTR when HIV-1 gene expression is minimal and that a threshold level of HIV-1 gene products is necessary for HCMV to inhibit this promoter.

DNA immunization confers protection against murine cytomegalovirus infection.

The murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) encodes an 89-kDa phosphoprotein (pp89) which plays a key role in protecting BALB/c mice against the lethal effects of the MCMV infection. In this report, we have addressed the question of whether "naked DNA" vaccination with a eukaryotic expression vector (pcDNA-89) that contains the MCMV IE1 gene driven by a strong enhancer/promoter can confer protection. BALB/c mice were immunized intradermally with pcDNA-89 or with the plasmid backbone pcDNAI/Amp (pcDNA) and then challenged 2 weeks later with either a lethal or a sublethal intraperitoneal dose of the K181 strain of MCMV. Variable results were obtained for the individual experiments in which mice received a lethal challenge. In four separate trials, an average of 63% of the mice immunized with pcDNA-89 survived, compared with 18% of the mice immunized with pcDNA. However, in two other trials there was no specific protection. The results of experiments in which mice were injected with a sublethal dose of MCMV were more consistent, and significant decreases in viral titer in the spleen and salivary glands of pcDNA-89-immunized mice were observed, relative to controls. At the time of peak viral replication, titers in the spleens of immunized mice were reduced 18- to >63-fold, while those in the salivary gland were reduced approximately 24- to 48-fold. Although DNA immunization elicited only a low level of seroconversion in these mice, by 7 weeks postimmunization the mice had generated a cytotoxic T-lymphocyte response against pp89. These results suggest that DNA vaccination with selected CMV genes may provide a safe and efficient means of immunizing against CMV disease.

Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins.

We have identified three open reading frames (ORFs) in murine cytomegalovirus (MCMV), designated M82, M83, and M84, which likely encode homologs of the human cytomegalovirus (HCMV) UL82 and UL83 matrix phosphoproteins. These ORFs, in the HindIII C fragment of MCMV, are colinear with the UL82, UL83, and UL84 ORFs of HCMV. M82 encodes a 598-amino-acid (aa) protein with homology to UL82, M83 encodes an 809-aa protein with homology to UL82 and UL83, and M84 encodes a 587-aa protein with homology to UL83 and UL84. Analysis of transcription by Northern (RNA) blotting indicated that the M82 and M83 ORFs are transcribed as 2.2- and 5-kb mRNAs, respectively, at 24 to 48 h postinfection (p.i.), while M84 is transcribed as a 6.9-kb mRNA only at 8 h p.i. All transcripts appear to terminate at the same position 3' of the M82 ORF. Of the products of the three ORFs, only M83 is strongly recognized by hyperimmune mouse serum. The M83 protein is a virion-associated phosphoprotein with an apparent molecular mass of 125 kDa. In MCMV-infected cells, it is detectable by Western blotting (immunoblotting) only at 48 h p.i. in the absence of phosphonoacetic acid, consistent with late gene expression. The M83 ORF is also expressed at high levels in cells infected by a recombinant vaccinia virus and yields a protein which is serologically cross-reactive and comigrates with the authentic MCMV protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

CREB and CREB-binding proteins play an important role in the IE2 86-kilodalton protein-mediated transactivation of the human cytomegalovirus 2.2-kilobase RNA promoter.

The human cytomegalovirus (HCMV) immediate-early region 2 86-kDa protein (IE2 86) is the major transactivator of the promoter for the 2.2-kb class of early RNAs (open reading frame UL 112-113). Previously, we reported that a DNA segment on this promoter between nucleotides (nt) -113 and -59 was critical for activation by IE2 86 in vivo and could be bound by IE2 86 in vitro (R. Schwartz, M. H. Sommer, A. Scully, and D. H. Spector, J. Virol. 68:5613-5622, 1994). With a set of site-specific mutations within nt -84 to -61, we have localized the essential cis-acting sequences to nt -72 to -61, which contain an ATF/CREB-binding site. The IE2 86-binding site between nt -113 and -85 is not essential for activation of the promoter by IE2 86 in transient-expression assays, but its presence can enhance the level of activation mediated through the sequences located between nt -84 and -59. Electrophoretic mobility shift assays with a segment containing nt -84 to -59 and nuclear extracts from human cells permissive for the HCMV infection revealed a complex band pattern. However, by supershift analysis with specific antibodies, we were able to identify CREB as the major ATF/CREB family member in the protein-DNA complexes. Further evidence that CREB is a target for IE2 86-mediated induction, is provided by the finding that IE2 86 activates the somatostatin promoter to high levels. Although the binding of IE2 86 to nonphosphorylated full-length CREB or deltaCREB is minimal, IE2 86 does form complexes with p300 and the CREB-binding protein (CBP), which in turn bind to CREB and can serve as adaptor proteins for CREB function. In addition, the in vivo functional relevance of the interaction between IE2 86 and CBP is indicated by the ability of IE2 86 to enhance transcriptional activation mediated by a GAL4-CBP fusion protein brought to a promoter by GAL4-binding sites.

Activation and regulation of human cytomegalovirus early genes.

The multiple pathogenic effects of human cytomegalovirus (HCMV) result from a complex interplay of viral gene products and induced and repressed cellular functions. HCMV immediate early and early gene expression clearly play a pivotal role in this scheme. I describe here work directed at elucidating the sequence requirements and trans-acting factors necessary for the activation and regulation of HCMV early genes. The focus is on three transcription units which are all activated at early times, but exemplify differential patterns of expression as the infection progresses, including: the major 2.7- and 1.2-kb transcripts encoded by the repeat bounding of the long unique segment of the genome, and a family of differentially spliced transcripts (originally designated the 2.2-kb RNAs-ORF UL112-113) which encode four proteins that are required in the transient complementation assay for HCMV DNA replication. Selected other early genes which illustrate alternative mechanisms of control are also discussed.

Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest.

Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect cellular proliferation, we investigated the impact of HCMV infection on key components of the cell cycle. Early after infection, HCMV induced elevated levels of cyclin E, cyclin E-associated kinase activity, and two tumor suppressor proteins, p53 and the retinoblastoma gene product (Rb). The steady-state concentration of Rb continued to rise throughout the infection, with most of the protein remaining in the highly phosphorylated form. At early times, HCMV infection also induced cyclin B accumulation, which was associated with a significant increase in mitosis-promoting factor activity as the infection progresses. In contrast, the levels of cyclin A and cyclin A-associated kinase activity increased only at late times in the infection, and the kinetics were delayed relative to those for cyclins E and B. Analysis of the cellular DNA content in the infected cells by flow cytometry showed a progressive shift of the cells from the G1 to the S and G2/M phases of the cell cycle, leading to an accumulation of aneuploid cells at late times. We propose that these HCMV-mediated perturbations result in cell cycle arrest in G2/M.