Human Cytomegalovirus Immediate Early Gene Expression Research Articles

Inhibition of human cytomegalovirus immediate-early gene expression and replication by the ethyl acetate (EtOAc) fraction of Elaeocarpus sylvestris in vitro

BackgroundIn immunocompromised patients, human cytomegalovirus (HCMV) infection can lead to severe, life-threatening diseases, such as pneumonitis, hepatitis, gastrointestinal tract disease, and retinitis. We previously reported that a 70% ethanol extract of Elaeocarpus sylvestris leaves (ESE) inhibits human cytomegalovirus (HCMV) replication in vitro. In the present study, we determined the solvent fraction of ESE that inhibits HCMV replication using activity-guided fractionation.MethodsActivity-guided fractionation of ESE was performed to determine the solvent fraction that inhibits HCMV replication. Effects of solvent fractions on HCMV lytic gene expression and major immediate-early (MIE) enhancer/promoter activity were further investigated.ResultsAmong the solvent fractions tested, the EtOAc fraction of ESE markedly reduced HCMV lytic gene expression and viral replication in vitro without exerting significant cytotoxic effects against human foreskin fibroblasts (HFF). Furthermore, the EtOAc fraction negatively affected HCMV MIE enhancer/promoter activity.ConclusionOur data collectively indicate that the EtOAc fraction of ESE contains active constituents that inhibit HCMV MIE enhancer/promoter activity and viral replication. The EtOAc fraction of ESE is a good source of novel drug candidates for treatment of HCMV-associated diseases.

LPS promotes a monocyte phenotype permissive for human cytomegalovirus immediate-early gene expression upon infection but not reactivation from latency

Human cytomegalovirus (HCMV) infection of myeloid cells is closely linked with the differentiation status of the cell. Haematopoietic progenitors and CD14+ monocytes are usually non-permissive for lytic gene expression which can lead to the establishment of latent infections. In contrast, differentiation to macrophage or dendritic cell (DC) phenotypes promotes viral reactivation or renders them permissive for lytic infection. The observation that high doses of Lipopolysaccharide (LPS) drove rapid monocyte differentiation in mice led us to investigate the response of human monocytes to HCMV following LPS stimulation in vitro. Here we report that LPS triggers a monocyte phenotype permissiveness for lytic infection directly correlating with LPS concentration. In contrast, addition of LPS directly to latently infected monocytes was not sufficient to trigger viral reactivation which is likely linked with the failure of the monocytes to differentiate to a DC phenotype. Interestingly, we observe that this effect on lytic infection of monocytes is transient, appears to be dependent on COX-2 activation and does not result in a full productive infection. Thus LPS stimulated monocytes are partially permissive lytic gene expression but did not have long term impact on monocyte identity regarding their differentiation and susceptibility for the full lytic cycle of HCMV.

Correction: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

Correction: Mitogen and Stress Activated Kinases Act Co-operatively with CREB during the Induction of Human Cytomegalovirus Immediate-Early Gene Expression from Latency

The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms.

Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. Importance: Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection.

Mitogen and stress activated kinases act co-operatively with CREB during the induction of human cytomegalovirus immediate-early gene expression from latency.

The devastating clinical consequences associated with human cytomegalovirus (HCMV) infection and reactivation underscores the importance of understanding triggers of HCMV reactivation in dendritic cells (DC). Here we show that ERK-mediated reactivation is dependent on the mitogen and stress activated kinase (MSK) family. Furthermore, this MSK mediated response is dependent on CREB binding to the viral major immediate early promoter (MIEP). Specifically, CREB binding to the MIEP provides the target for MSK recruitment. Importantly, MSK mediated phosphorylation of histone H3 is required to promote histone de-methylation and the subsequent exit of HCMV from latency. Taken together, these data suggest that CREB binding to the MIEP is necessary for the recruitment of the kinase activity of MSKs to initiate the chromatin remodelling at the MIEP required for reactivation. Thus the importance of CREB during HCMV reactivation is to promote chromatin modifications conducive for viral gene expression as well as acting as a classical transcription factor. Clearly, specific inhibition of this interaction between CREB and MSKs could provide a strategy for therapeutic intervention.

The extract of Elaeocarpus sylvestris inhibits human cytomegalovirus immediate early gene expression and replication in vitro

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in newborn infants, immunocompromised individuals with HIV/AIDS and organ transplant recipients. In order to identify a novel antiviral candidate for HCMV-related diseases, crude ethanol extracts from plants were screened for their potential inhibitory activity on HCMV replication in vitro. Ethanol (70%) extract of Elaeocarpus sylvestris leaves (ESE) markedly inhibited the replication of the HCMV Towne strain without exhibiting any significant adverse effects on the viability of human foreskin fibroblasts (HFF). In addition, ESE significantly downregulated HCMV immediate early (IE) gene expression. Taken together, this is the first study, to the best of our knowledge, demonstrating that ESE has a potent antiviral activity against HCMV by downregulating HCMV IE gene expression and replication.

Inhibition of human cytomegalovirus IE gene expression by the extract of Glycyrrhiza uralensis

The human cytomegalovirus (CMV) preferentially infects tumor tissues, and the accumulated CMV immediate early (IE) antigen may lead to tumor promotion and progression. The development of strategies to inhibit the expression and/or function of human IE antigen is an important goal to prevent and treat certain forms of cancer associated with human CMV.

Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

Human cytomegalovirus immediate-early gene expression is restricted by the nuclear domain 10 component Sp100

Nuclear domains 10 (ND10s) are discrete subnuclear structures that contain the three major protein components promyelocytic leukaemia protein (PML), hDaxx and Sp100. Previous studies identified the ND10-components PML and hDaxx as cellular restriction factors that independently counteract human cytomegalovirus (HCMV) infection via the repression of viral immediate-early (IE) gene expression. Consequently, we asked whether Sp100 is likewise involved in this repressive activity. Infection of Sp100 knockdown (kd) cells with HCMV resulted in a significantly increased plaque-forming ability. In addition, ablation of Sp100 led to a considerable increase in the number of IE1-expressing cells, indicating that Sp100 suppresses the initiation of viral gene expression. Next, double-kd cells, lacking either Sp100/hDaxx or Sp100/PML, were generated. Here, infection resulted in an additional enhancement in HCMV replication efficacy compared with the single-kd cells. Thus, our results further strengthen the concept that the three major ND10-components independently contribute to the cellular restriction of HCMV replication.

Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

Knockdown of hDaxx in normally non-permissive undifferentiated cells does not permit human cytomegalovirus immediate-early gene expression

The cellular protein human Daxx (hDaxx), a component of nuclear domain 10 structures, is known to mediate transcriptional repression of human cytomegalovirus immediate-early (IE) gene expression upon infection of permissive cell types, at least in part, by regulation of chromatin structure around the major IE promoter (MIEP). As it is now clear that differentiation-dependent regulation of the MIEP also plays a pivotal role in the control of latency and reactivation, we asked whether hDaxx-mediated repression is involved in differentiation-dependent MIEP regulation. We show that downregulation of hDaxx by using small interfering RNA technology in undifferentiated NT2D1 cells does not permit expression of viral IE genes, nor does it result in changes in chromatin structure around the MIEP. Viral IE gene expression is only observed upon cellular differentiation, suggesting little involvement of hDaxx in the regulation of the viral MIEP in undifferentiated cells.

Nuclear domain 10 components promyelocytic leukemia protein and hDaxx independently contribute to an intrinsic antiviral defense against human cytomegalovirus infection.

Infection with DNA viruses commonly results in the association of viral genomes with a cellular subnuclear structure known as nuclear domain 10 (ND10). Recent studies demonstrated that individual ND10 components, like hDaxx or promyelocytic leukemia protein (PML), mediate an intrinsic immune response against human cytomegalovirus (HCMV) infection, strengthening the assumption that ND10 components are part of a cellular antiviral defense mechanism. In order to further define the role of hDaxx and PML for HCMV replication, we generated either primary human fibroblasts with a stable, individual knockdown of PML or hDaxx (PML-kd and hDaxx-kd, respectively) or cells exhibiting a double knockdown. Comparative analysis of HCMV replication in PML-kd or hDaxx-kd cells revealed that immediate-early (IE) gene expression increased to a similar extent, regardless of which ND10 constituent was depleted. Since a loss of PML, the defining component of ND10, results in a dispersal of the entire nuclear substructure, the increased replication efficacy of HCMV in PML-kd cells could be a consequence of the dissociation of the repressor protein hDaxx from its optimal subnuclear localization. However, experiments using three different recombinant HCMVs revealed a differential growth complementation in PML-kd versus hDaxx-kd cells, strongly arguing for an independent involvement in suppressing HCMV replication. Furthermore, infection experiments using double-knockdown cells devoid of both PML and hDaxx illustrated an additional enhancement in the replication efficacy of HCMV compared to the single-knockdown cells. Taken together, our data indicate that both proteins, PML and hDaxx, mediate an intrinsic immune response against HCMV infection by contributing independently to the silencing of HCMV IE gene expression.

Human Cytomegalovirus Gene Expression Is Silenced by Daxx-Mediated Intrinsic Immune Defense in Model Latent Infections Established In Vitro

In addition to productive lytic infections, herpesviruses such as human cytomegalovirus (HCMV) establish a reservoir of latently infected cells that permit lifelong colonization of the host. When latency is established, the viral immediate-early (IE) genes that initiate the lytic replication cycle are not expressed. HCMV IE gene expression at the start of a lytic infection is facilitated by the viral pp71 protein, which is delivered to cells by infectious viral particles. pp71 neutralizes the Daxx-mediated cellular intrinsic immune defense that silences IE gene expression by generating a repressive chromatin structure on the viral major IE promoter (MIEP). In naturally latently infected cells and in cells latently infected in vitro, the MIEP also adopts a similar silenced chromatin structure. Here we analyze the role of Daxx in quiescent HCMV infections in vitro that mimic some, but not all, of the characteristics of natural latency. We show that in these "latent-like" infections, the Daxx-mediated defense that represses viral gene expression is not disabled because pp71 and Daxx localize to different cellular compartments. We demonstrate that Daxx is required to establish quiescent HCMV infections in vitro because in cells that would normally foster the establishment of these latent-like infections, the loss of Daxx causes the lytic replication cycle to be initiated. Importantly, the lytic cycle is inefficiently completed, which results in an abortive infection. Our work demonstrates that, in certain cell types, HCMV must silence its own gene expression to establish quiescence and prevent abortive infection and that the virus usurps a Daxx-mediated cellular intrinsic immune defense mechanism to do so. This identifies Daxx as one of the likely multiple viral and cellular determinants in the pathway of HCMV quiescence in vitro, and perhaps in natural latent infections as well.

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.

Role of the cellular protein hDaxx in human cytomegalovirus immediate-early gene expression

Human cytomegalovirus (HCMV) immediate-early (IE) transcription is stimulated by virion phosphoprotein pp71, the product of gene UL82. It has previously been shown that pp71 interacts with the cellular protein hDaxx and, in the studies presented here, the significance of this interaction was investigated for HCMV IE gene expression. In co-transfection experiments, the presence of hDaxx increased the transcriptional response of the HCMV major IE promoter (MIEP) to pp71, but it was not possible to determine whether the effect was due to an interaction between the two proteins or to stimulation of hDaxx synthesis by pp71. The use of small interfering RNA (siRNA) in long- and short-term transfection approaches reduced intracellular hDaxx levels to no more than 3 % of normal. Infection of hDaxx-depleted cells with herpes simplex virus recombinants containing the HCMV MIEP revealed significantly greater promoter activity when hDaxx levels were minimal. Similarly, reducing intracellular hDaxx amounts resulted in greater IE gene expression during infection with an HCMV mutant lacking pp71, but had no effect on IE transcription during infection with wild-type HCMV. The results suggest that hDaxx is not important as a positive-acting factor for the stimulation of HCMV IE transcription by pp71. Instead, it appears that hDaxx acts as a repressor of IE gene expression, and it is proposed here that the interaction of pp71 with hDaxx is important to relieve repression and permit efficient initiation of productive replication.

Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells

Previous work from this laboratory has shown that expression of human cytomegalovirus (HCMV) immediate-early (IE) genes from the major immediate-early promoter (MIEP) is likely to be regulated by chromatin remodelling around the promoter affecting the acetylation state of core histone tails. The HCMV MIEP contains sequences that bind cellular transcription factors responsible for its negative regulation in undifferentiated, non-permissive cells. Ets-2 repressor factor (ERF) is one such factor that binds to such sequences and represses IE gene expression. Although it is not known how cellular transcription factors such as ERF mediate transcriptional repression of the MIEP, it is likely to involve differentiation-specific co-factors. In this study, the mechanism by which ERF represses HCMV IE gene expression was analysed. ERF physically interacts with the histone deacetylase, HDAC1, both in vitro and in vivo and this physical interaction between ERF and HDAC1 mediates repression of the MIEP. This suggests that silencing of viral IE gene expression, associated with histone deacetylation events around the MIEP, is mediated by differentiation-dependent cellular factors such as ERF, which specifically recruit chromatin remodellers to the MIEP in non-permissive cells.

Thrombin induces Sp1-mediated antiviral effects in cytomegalovirus-infected human retinal pigment epithelial cells

Human cytomegalovirus (HCMV) retinitis causing retinal detachment and destruction of the blood-retina barrier is closely related to retinal hemorrhage/coagulation. However, the effects of procoagulants on HCMV (re)activation in retinal cells have not been investigated yet. Therefore, we studied whether thrombin modulates the expression of HCMV immediate early (IE) and late (L) genes in cultured human retinal pigment epithelial cells (RPE). Thrombin specifically stimulated the protease-activated receptor-1 (PAR-1) on RPE and, surprisingly, inhibited basal and 12,0-tetradecanoylphorbol 13-acetate-stimulated HCMV IE gene expression in infected RPE. On the other hand, HCMV strongly induced Sp1 DNA binding activity, which was prevented by thrombin/PAR1-mediated Sp1 hyperphosphorylation. Our data suggest that thrombin/PAR-1 may inhibit Sp1-dependent HCMV replication, which might be an important regulatory mechanism for HCMV persistence and replication in RPE.

Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection.

We have used horseradish peroxidase-labeled 40 mer oligodeoxynucleotides (HRP-ODNs) specific for the human cytomegalovirus immediate early gene (HCMV-IE) and a novel dinitrophenol-tyramide signal amplification reagent (DNP-TSA plus) to evaluate their utility in fluorescence in situ hybridization (FISH). For DNA FISH, single or cocktails of HRP-ODNs were hybridized to metaphase chromosomes of rat 9G cells which, as determined by DNA fiber FISH, carry an integrated tandem repeat of 50-60 copies of the HCMV-IE gene. With one layer of DNP-TSA plus deposition and subsequent detection with a fluorochrome-conjugated antibody, four HRP-ODNs were needed to detect the HCMV-IE integration site. When employing two TSA amplification rounds, one HRP-ODN was sufficient for obtaining a strong signal of the integrated gene cluster, indicating that 50-60 HRP molecules can be detected with ease. In addition to DNA FISH, we report here the first use of HRP-ODN probes for mRNA detection by FISH. A single HRP-ODN and one DNP-TSA plus step resulted in clear visualization of the HCMV-IE gene transcripts in rat 9G cells induced for HCMV-IE expression by cycloheximide. Two TSA detection steps enhanced signal intensities even further. Parallel experiments with hapten-labeled ODN and cDNA probes and conventional detection methods illustrated the superiority of the HRP-ODN/TSA approach in DNA and RNA FISH.

Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

Regulation of human cytomegalovirus immediate-early gene expression.

The positive and negative cis-acting elements that affect transcription from the human cytomegalovirus major immediate-early (MIE) promoter and the viral and cellular proteins that bind to these elements are discussed. The data obtained using in vitro transcription and transient transfection assays are reviewed and compared to recent data using recombinant viruses with cis-acting elements deleted. The effects of cell type and cellular differentiation on activation of transcription from the MIE promoter are compared with the effects of mitogens and virion-associated tegument proteins that directly or indirectly activate protein kinase pathways. The repressor and enhancer regions upstream of the MIE promoter in the large unique component of the viral genome are compared to the elements upstream of the more simplified IEUS3 promoter in the short unique component of the viral genome.