Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-3 and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.     

Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production

The effect of human cytomegalovirus (HCMV) gene expression on beta interferon (IFN-beta) expression was examined. We demonstrate that the HCMV immediate-early 2 (IE2) gene product IE86 can effectively block the induction of IFN-beta during HCMV infection. IE86 also efficiently blocked the induction of IFN-beta following Sendai virus infection, demonstrating that IE86's ability to block induction of IFN-beta is not limited to HCMV infection, identifying IE2 as an IFN-beta antagonist.     

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.     

Inhibition of Human Cytomegalovirus Infection by IE86-Specific Short Hairpin RNA-Mediated RNA Interference

To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.     

Development of cell lines that provide tightly controlled temporal translation of the human cytomegalovirus IE2 proteins for complementation and functional analyses of growth-impaired and nonviable IE2 mutant viruses

The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.     

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.     

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.     

Human cytomegalovirus (HCMV) UL82 gene product (pp71) relieves hDaxx-mediated repression of HCMV replication

This study examines the role of the cellular protein hDaxx in controlling human cytomegalovirus (HCMV) immediate-early (IE) gene expression and viral replication. Using permissive cell lines that either overexpress hDaxx or are depleted of hDaxx expression by the use of short hairpin RNA, we demonstrate that hDaxx functions as a repressor of HCMV IE gene expression and replication. In addition, we demonstrate that the impaired growth phenotype associated with the UL82 (pp71) deletion mutant is abolished when hDaxx knockdown cells are infected, suggesting that pp71 functions to relieve hDaxx-mediated repression during HCMV infection.     

The phosphorylation status of the serine-rich region of the human cytomegalovirus 86-kilodalton major immediate-early protein IE2/IEP86 affects temporal viral gene expression

The 86-kDa major immediate-early protein (IE2/IEP86) of human cytomegalovirus (HCMV) contains a serine-rich region (amino acids 258 to 275) with several consensus casein kinase II (CKII) sites. We performed extensive mutational analysis of this region, changing serines to alternating alanines and glycines. Mutation of the serines between amino acids 266 and 275 eliminated in vitro phosphorylation by CKII. In vitro CKII phosphorylation of the serines between amino acids 266 and 269 or between amino acids 271 and 275 inhibited the ability of IE2/IEP86 to bind to TATA-binding protein. Correspondingly, nonphosphorylatable mutants in these regions showed increased activation of specific HCMV gene promoters in transfection studies. Viruses containing mutations of the serines throughout the entire region (amino acids 258 to 275) or the second half (amino acids 266 to 275) of the region showed delayed expression of all viral proteins tested and, correspondingly, delayed growth compared to wild-type HCMV. Mutation of the serines in the first half of the serine-rich region (amino acids 258 to 264) or between amino acids 266 and 269 propagated very slowly and has not been further studied. In contrast, mutation of the serines between amino acids 271 and 275 resulted in accelerated virus growth and accelerated temporal expression of viral proteins. These results suggest that the serine-rich region is structurally complex, possibly affecting multiple functions of IE2/IEP86. The data show that the phosphorylation state of the serine-rich region, particularly between amino acids 271 and 275, modulates the temporal expression of viral genes.     

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.     

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.