CpG methylation in mammalian DNA is known to interfere with gene expression by inhibiting the binding of transactivators to their cognate sequence motifs or recruiting proteins involved in gene repression. An Epstein–Barr virus-encoded transcription factor, Zta, was the first example of a sequence-specific transcription factor that preferentially recognizes and selectively binds DNA sequence motifs with methylated CpG residues, reverses epigenetic silencing and activates gene transcription. The DNA binding domain of Zta is homologous to c-Fos, a member of the cellular AP-1 (activator protein 1) transcription factor family, which regulates cell proliferation and survival, apoptosis, transformation and oncogenesis. We have identified a novel AP-1 binding site termed meAP-1, which contains a CpG dinucleotide. If methylated, meAP-1 sites are preferentially bound by the AP-1 heterodimer c-Jun/c-Fos in vitro and in cellular chromatin in vivo. In activated human primary B cells, c-Jun/c-Fos locates to these methylated elements in promoter regions of transcriptionally activated genes. Reminiscent of the viral Zta protein, c-Jun/c-Fos is the first identified cellular member of the AP-1 family of transactivators that can induce expression of genes with methylated, hence repressed promoters, reversing epigenetic silencing.

The human herpesviruses Epstein-Barr virus (EBV) and, supporting an essential role of EBV in HL lymphomagenesis (1). EBV ϩ HRS cells express the viral protein latent membrane protein 2A (LMP2A), which can functionally replace the BCR because Ig-negative B cells can only survive if infected with EBV but not with an LMP2A-negative EBV mutant (4). BCR and LMP2A both contain an immunoreceptor tyrosine-based activation motif (ITAM) (5, 6), which is central for the induction of BCR signaling cascades. In contrast to the BCR, LMP2A is thought to be constitutively active and independent of any ligand or antigen encounter (references 7 and 8 and references therein). EBV also encodes LMP1, a constitutive CD40 receptor mimic that provides a second survival signal for B cells. LMP1 together with LMP2A activates HRS cells, protects them from apoptosis, and induces their proliferation (1, 9).PEL tumor cells are infected with KSHV, and ca. 80% are EBV ϩ , although LMP1 and LMP2A are barely expressed (10,11

The major immediate-early (MIE) genes of cytomegaloviruses (CMV) are broadly thought to be decisive regulators of lytic replication and reactivation from latency. To directly assess the role of the MIE protein IE1 during the infection of murine CMV (MCMV), we constructed an MCMV with exon 4 of the ie1 gene deleted. We found that, independent of the multiplicity of infection, the resulting recombinant virus, MCMVdie1, which fails to express the IE1 protein, was fully competent for early gene expression and replicated in different cultured cell types with identical kinetics to those of parental or revertant virus. Immunofluorescence microscopy studies revealed that MCMVdie1 was greatly impaired in its capacity to disrupt promyelocytic leukemia bodies in NIH 3T3 cells early after infection, a process that has been proposed to increase viral transcription efficiency. We examined MCMVdie1 in the murine model using both immunocompetent BALB/c and severe combined immunodeficient (SCID) mice. When MCMVdie1 was inoculated into these two types of mice, significantly lower viral titers were detected in infected organs than in those of the wild-type virus-infected animals. Moreover, the ie1-deficient MCMV exhibited a markedly reduced virulence. While all animals infected with 5 ؋ 10 4 PFU of parental virus died by 30 days postinfection, SCID mice infected with a similar dose of MCMVdie1 did not succumb before 60 days postinfection. The in vivo defective growth phenotype of MCMVdie1 was abrogated upon rescue of ie1. These results demonstrate the significance of the ie1 gene for promoting an acute MCMV infection and virulence yet indicate that MCMV is able to grow in vivo, although impaired, in the absence of the ie1 gene.Similar to other herpesviruses, the transcription of the cytomegalovirus (CMV) genome during the lytic infection is temporarily regulated (for a review, see reference 47). The immediate-early (IE or ␣) genes are the first ones to be expressed in the replicative cycle, and their expression does not depend on prior viral protein synthesis. Together with some virion proteins, the IE products activate viral genes and alter the infected cell to generate an appropriate milieu that favors viral replication. Transcription of early (E or ␤) genes requires the expression of at least one of the IE proteins, and only after viral replication has started, the transcription of late (L or ␥) genes proceeds. The majority of the CMV IE transcripts originate from the major IE (MIE) locus. This locus is structurally similar between human CMV (HCMV) and the closely related mouse CMV (MCMV) (14,53,59). The primary transcript from the MIE region is under the control of the strong MIE enhancer-promoter and is differentially spliced to generate two predominant transcripts, the ie1 transcript that consists of exons 1 to 4, and the ie2 transcript that is composed of exons 1 to 3 and 5. In HCMV, the ie1 and ie2 transcripts are translated into the acidic 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins, respectively (for a review, ...

The role of NF-B in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-B response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEPenhancer that, although this region is essential for HCMV growth, none of the four NF-B response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.The major immediate-early promoter (MIEP) of human cytomegalovirus (HCMV) is responsive to a multitude of transcription factors and plays a pivotal role in initiating the viral transcription/replication cycle (7, 16; reviewed in references 22 and 23). Regulation of the MIEP has been postulated to be critical in determining HCMV permissiveness and the transition between latent and lytic infection. Thus, deciphering the molecular mechanisms of the MIEP regulation may reveal key control points contributing to HCMV pathogenesis.The MIEP enhancer includes four cognate NF-B recognition sites, and NF-B activates MIEP transcription in transient-transfection assays (20,(25)(26)(27). HCMV infection results in rapid induction of cellular 27,30), and several groups have reported a potential contribution of NF-B to the replication strategy of HCMV through regulation of the MIEP (8, 13). In contrast, we and others have reported a neutral or even a negative role of NF-B activation on HCMV transcription/replication cycle in different cell types (3,4,11,14,15). However, the basis for these experimental discrepancies is currently unclear. Importantly, a direct test of the requirement for the MIEP NF-B binding sites in HCMV transcription/ replication has still not been performed. Here we report on formally assessing the direct requirement of the cognate binding sites for NF-B in contributing to major immediate-early (MIE) transcription and viral growth.As a first step toward understanding NF-B regulation of the HCMV MIEP, we deleted enhancer sequences from Ϫ52 to Ϫ667 (including all NF-B response elements), in HCMV AD169. A parental HCMV bacterial artificial chromosome (BAC) (5, 6) containing the E-GFP open reading frame (ORF) under control of the murine cytomegalovirus (MCMV) MIEP (Fig. 1A, line 1) was used to construct two enhancerless HCMV recombinant mutants. In HCMVdE, MIEP sequences from Ϫ52 to Ϫ667 were removed (Fig. 1A, line 2), and in HCMVdE::Kan, enhancer sequences were replaced with a 1-kbp stuffer region to maintain the genomic spatial integrity of the ie1/ie2 and UL127 promoters (Fig. 1A, line 3). Once the integrity of constructed HCMV genomes was confirmed by restriction analysis (data not shown), they were transfected in MRC-5 fibroblasts. Three days posttransfection, ϳ100 single cells expressing green fluorescent prot...

Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Human cytomegalovirus (HCMV) replication begins with the expression of the major immediate-early (MIE) gene products IE1 and IE2, which are multifunctional proteins mainly involved in regulating both viral and cellular gene expression (reviewed in reference 51). The MIE proteins are essential for the progression of the replication cycle and crucial determinants of the transition from latency to reactivation (62, 63). Hence, the regulation of their expression is a key point in controlling the outcome of the HCMV infectious programs.Transcription of the HCMV MIE genes is driven by a complex and potent promoter, the MIE promoter (MIEP), which comprises different functional units including a basal promoter, the enhancer region, and the modulator (23). The MIEP contains binding sites for a diverse set of signal-regulated stimulatory and inhibitory transcription factors, such as NF-B, ATF/CREB, activator protein 1 (AP-1), YY1, Sp1/ Sp3, and retinoic acid receptor (RAR)/retinoid X receptor (RXR), most of them densely packed in the enhancer region (48). In addition, viral tegument proteins and the MIE proteins themselves have also been shown to modulate MIEP activity. During latency, the MIEP is associated with markers of repressed heterochromatin, remaining silent (53, 59). Cellular differentiation and alterations in the levels of specific transcription factors by a variety of stimuli promote the activation of the MIEP and thereby the expression of downstream MIE genes. Consequently, MIEP activity is dependent on cell type, cellular differentiation stage, and the activity of specific signaling transduction pathways. Work with transgenic mice carrying a LacZ reporter under the control of the HCMV MIEP enhancer indicated that the expression of the MIEP is restricted to specific cell types in multiple organs, paralleling tissues normally infected by HCMV in the natural host (5,6,41). A number of studies in the last several years have addressed the relevance of different segments of the MIEP for MIE gene expression and viral replication (27,33,34,47,49). While the more distal component of the enhancer (spanning from Ϫ550 to Ϫ300 relative to the transcription start site [ϩ1] of the MIEP) has been shown only to partially contribute to viral replication at a low multiplicity of infection (MOI) (47), progressive deletions starting from the distal end of the proximal segment of the enhancer (spanning from Ϫ300 to Ϫ39) resulted in recombinant viruses that replicated slower and with

The human cytomegalovirus (HCMV) major immediate-early enhancer has been postulated to play a pivotal role in the control of latency and reactivation. However, the absence of an animal model has obstructed a direct test of this hypothesis. Here we report on the establishment of an in vivo, experimentally tractable system for quantitatively investigating physiological functions of the HCMV enhancer. Using a neonate BALB/c mouse model, we show that a chimeric murine CMV under the control of the HCMV enhancer is competent in vivo, replicating in key organs of mice with acute CMV infection and exhibiting latency/reactivation features comparable for the most part to those of the parental and revertant viruses.Reactivation of human cytomegalovirus (HCMV) in an immunocompromised host frequently leads to a variety of severe complications, such as pneumonia, hepatitis, and retinitis (30). The HCMV major immediate-early promoter (MIEP) has been regarded as a key genetic element in determining the commencement of lytic infection and the switch from latency to reactivation (17,35,36). The MIEP steers the extent and patterns of expression of the MIE genes, which encode multifunctional proteins required for the productive replication cycle (25, 28). The enhancer region of the MIEP is controlled by a complex interplay between host factors and virally encoded proteins (12, 36). Thus, binding sites for multiple signal-regulated cellular factors, such as NF-B, CREB/ATF, Sp1, AP-1, YY1, Ets, RAR/RXR, and serum response factors, lie in this regulatory region. The importance of the HCMV MIEP enhancer in the context of the infection of cultured cells has been documented (15,18,20,26,27). However, the lack of an animal model system that sustains significant HCMV replication has prevented the assessment of the role and mechanisms of action of this region during latent infection. Thus, there is an urgent need to develop in vivo models to address this issue.Infection of mice with murine CMV (MCMV) has proven to be an invaluable model for studying aspects of the biology of CMV infection. The MCMV MIE locus resembles in many ways its HCMV counterpart, and significant information has been drawn from this system concerning MIE gene functions and MIEP regulation (8,33). In this context, we have described the absolute requirement of the MCMV enhancer for productive infection in its natural host (13). While the primary sequence and architecture of the MCMV and HCMV enhancers are quite different, they share many of the same signal-regulatory control elements (7, 10, 12), conferring both similar and distinct biological properties to them. Accordingly, the first attempts to study HCMV MIEP function in an intact physiological system involved developing murine transgenic models using an HCMV enhancer linked to a reporter gene (3,4,23). However, while informative, these models place the enhancer out of its natural environment of the viral genome and most importantly out of the context of an infection. For these reasons, we sought to address HCMV-enha...

In infected cells, Epstein–Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA’s transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

Over the last decade, INFRAFRONTIER has positioned itself as a world-class Research Infrastructure for the generation, phenotyping, archiving, and distribution of mouse models in Europe. The INFRAFRONTIER network consists of 22 partners from 15 countries, and is continuously enhancing and broadening its portfolio of resources and services that are offered to the research community on a non-profit basis. By bringing together European rodent model expertise and providing valuable disease model services to the biomedical research community, INFRAFRONTIER strives to push the accessibility of cutting-edge human disease modelling technologies across the European research landscape. This article highlights the latest INFRAFRONTIER developments and informs the research community about its extensively utilised services, resources, and technical developments, specifically the intricacies of the INFRAFRONTIER database, use of Curated Disease Models, overview of the INFRAFRONTIER Cancer and Rare Disease resources, and information about its main state-of-the-art services. Graphical abstract