Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.     

Modulation of T-cell activation through protein kinase C- or A-dependent signalling pathways synergistically increases human immunodeficiency virus long terminal repeat induction by cytomegalovirus immediate-early proteins.

By using human CD4+ lymphoblastoid T cells transiently cotransfected with human immunodeficiency virus (HIV) and cytomegalovirus (CMV), we tested whether modulation of T-cell activation through the protein kinase C (PKC) or the protein kinase A (PKA) pathway synergized with CMV immediate-early (IE) proteins in HIV long terminal repeat (LTR) transactivation. Stimulation with phorbol myristate acetate, tumor necrosis factor, or cross-linked antibodies to CD3 and CD28 resulted in modest enhancement (two- to fourfold) of the activity of a luciferase expression vector under control of the HIV LTR. Cotransfection of a vector expressing the CMV IE1 and IE2 proteins under the control of their own promoter enhanced HIV LTR activity 16- to 49-fold. Combination of any one of the above stimuli and CMV IE expression amplified HIV LTR activity 99- to 624-fold. Stimulation of PKA-dependent pathways with forskolin, 8-bromo cyclic AMP, or prostaglandin E2 had a minimal effect on HIV LTR activity, whereas such stimuli resulted in synergistic amplification in cells cotransfected with CMV IE (three- to fivefold increases over the effects of CMV IE alone). This synergism was independent of the NF-kappa B binding motifs within the HIV LTR. CMV IE2, but not IE1, protein induced HIV transactivation and synergized with signals modulating T-cell activation. The intense synergism observed was superior to the increase in IE protein expression following PKC activation by phorbol myristate acetate. Treatment of cells with PKC inhibitor GF109203X blocked most of the observed synergism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b

The 86-kDa IE2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a potent transactivator of viral as well as cellular promoters. Several lines of evidence indicate that this broad transactivation spectrum is mediated by protein-protein interactions. To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the isolation of two ubiquitin-homologous proteins, SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2. The polypeptides SUMO-1 and hSMT3b have previously been shown to be covalently coupled to a subset of nuclear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By Western blot analysis, we were able to show that the IE2-p86 protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1. Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling. By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant. This suggests a functional relevance of covalent modification by ubiquitin-homologous proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.     

The human cytomegalovirus IE2 86-kilodalton protein interacts with an early gene promoter via site-specific DNA binding and protein-protein associations.

The 86-kDa immediate-early 2 protein (IE2 86) of human cytomegalovirus is a powerful transactivator of homologous and heterologous promoters, including the human cytomegalovirus 1.2-kb RNA early promoter. Two potential mechanisms for gene activation by IE2 86 include interaction with cellular proteins and direct DNA binding. In this report, we show that the 1.2-kb RNA promoter contains a cis-acting AP-1 site, critical for its activation by IE2 86 in vivo, and that IE2 86, purified as a glutathione S-transferase-IE86 fusion protein, can interact with c-Jun and JunB. Additionally, by coimmunoprecipitation, we document that JunB and IE2 86 do associate in vivo. Further in vitro analysis reveals that Fos proteins are able to associate with glutathione S-transferase-IE86 only when present as a Jun-Fos heterodimer. With a set of IE2 86 mutants, we demonstrate that three independent regions of the IE2 86 interact in vitro with c-Jun, two of which are essential for activation of the 1.2-kb RNA promoter in vivo. We also show that IE2 86 can bind directly to this promoter through a sequence located just upstream of the AP-1 site between nucleotides -125 and -97. This discrete domain shares sequence homology with the cis-repression signal on the IE gene.     

Effect of Inducible Expressed Human Cytomegalovirus Immediate Early 86 Protein on Cell Apoptosis

Human cytomegalovirus is a common human pathogen that can cause life-threatening disease under certain conditions. During infection of host cells, the virus expresses regulatory proteins such as IE72 and IE86 that are important for viral propagation. IE86 plays a critical role in the modulation of viral replication as well as host cell cycle control and apoptosis. In this study, a Tet-On system was used to quantify the effect of IE86 on apoptosis and p53 expression. Our results indicate that IE86 inhibits tumor necrosis factor (TNF)-α induced apoptosis and that the anti-apoptotic activity of this viral protein correlates with its expression levels. In addition, IE86 did not alter the mRNA level of p53. The system developed should provide a method for functional analysis of human cytomegalovirus (HCMV) IE86 protein.     

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.     

A 10-base-pair element of the human immunodeficiency virus type 1 long terminal repeat (LTR) is an absolute requirement for transactivation by the human cytomegalovirus 72-kilodalton IE1 protein but can be compensated for by other LTR regions in transactivation by the 80-kilodalton IE2 protein.

Transient gene expression studies have indicated that human cytomegalovirus (HCMV) specifically transactivates the human immunodeficiency virus (HIV) long terminal repeat (LTR). We show here, by a specific mutational analysis, that only the TATA box region is obligatory for transactivation of the HIV-1 LTR by HCMV. Similarly, this element is also sufficient for transactivation by either the HCMV 72-kDa major immediate-early 1 (IE1) or 80-kDa IE2 gene product independently. However, deletion of a 10-bp region from the minimal responsive element, 5' to the TATA box, dramatically reduced the level of HCMV 72-kDa IE1 or 80-kDa IE2 transactivation, indicating a crucial role for this element in transactivation. Whereas inclusion of the TAR element or Sp1 sites on this 10-bp-deleted minimal promoter had no effect on the removal of IE1 transactivation, TAR and Sp1 elements did compensate for the 10-bp element in transactivation by IE2 and HCMV. Consequently, the sequence requirements of the HIV-1 LTR for transactivation by HCMV can be reproduced by these IE1 and IE2 gene products of HCMV.     

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.     

Role of human cytomegalovirus immediate-early proteins in cell growth control

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G(2)/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.     

Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells.

Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).     

CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein.

Human cytomegalovirus (HCMV) immediate-early (IE) proteins are known potent transregulators of viral and cellular gene expression upon HCMV infection. HCMV is known to activate a number of cellular genes intimately associated with the cell cycle and DNA replication by mechanisms involving the viral major IE 86-kDa protein (IE2). We have recently shown that IE2 mediates this activation in a TATA-dependent manner and interacts directly with the TATA-binding protein. However, a number of TATA-less cellular promoters, e.g., DNA polymerase alpha and dihydrofolate reductase, are also activated by HCMV infection. Consequently, we have asked how HCMV mediates this activation. We show that, consistent with its known TATA dependency, IE2 does not activate the DNA polymerase alpha promoter. In contrast, this promoter is strongly activated by the major IE 72-kDa protein (IE1). Whilst deletion of ATF or E2F sites within the DNA polymerase alpha promoter had little effect on IE1-mediated activation, removal of the CCAAT box appeared to abolish high levels of activation by IE1. Consistent with this observation, we also find that IE1 interacts directly with the CCAAT box binding factor CTF1 in vitro and massively augments CTF1-mediated activation of the DNA polymerase alpha promoter in transient transfection assays.