No abstract

The repression of human cytomegalovirus immediate-early (IE) lytic gene expression is crucial for the maintenance of the latent viral state. By using conditionally permissive cell lines, which provide a good model for the differentiation state-dependent repression of IE gene expression, we have identified several cellular factors that bind to the major immediate-early promoter (MIEP) and whose expression is down-regulated after differentiation to a permissive phenotype. Here we show that the cellular protein Ets-2 Repressor Factor (ERF) physically interacts with the MIEP and represses MIEP activity in undifferentiated non-permissive T2 embryonal carcinoma cells. This factor binds to the dyad element and the 21 bp repeats within the MIEP -regions known to be important for the negative regulation of MIEP activity. Finally, we show that following differentiation to a permissive phenotype ERF's repressive effects are severely abrogated. INTRODUCTIONHuman cytomegalovirus (HCMV) is a betaherpesvirus whose sero-prevalence can vary from 50 to 90 % depending on the socio-economic status of the population. Following primary infection, which rarely causes disease in the immunocompetent, HCMV maintains a latent infection throughout the lifetime of the infected host. However, infection or reactivation in the immunocompromised, such as transplant recipients or AIDS patients, is associated with morbidity and mortality (reviewed in Alford & Britt, 1993).Productive HCMV infection and reactivation require an ordered cascade of gene expression and are dependent on the expression of the immediate-early (IE) gene products IE72 and IE86 (also known as IE1 and IE2 respectively). These IE proteins are potent and promiscuous transactivators of both cellular and viral genes, and serve to activate the viral early (E) gene promoters (Pizzorno et al., 1988;Malone et al., 1990), ultimately leading to viral DNA replication, expression of the viral late (L) genes and virus assembly. The expression of IE72 and IE86 is under the control of the major immediate-early promoter (MIEP), a highly complex region comprising a TATA box, an upstream imperfect dyad symmetry element (also termed the modulator), and an extremely powerful enhancer region made up of a series of 17, 18, 19 and 21 bp repeat motifs (reviewed in Meier & Stinski, 1996). MIEP activity is regulated by a myriad of positively and negatively acting cellular and viral proteins. For example, there are numerous nuclear factor-1 (NF-1) sites within the MIEP and the 18 and 19 bp repeat sequences contain NF-kB and cyclic AMPresponsive element binding protein (CREB) binding sites respectively (Meier & Stinski, 1996). Conversely, several studies using non-permissive cells have shown that the modulator and the 21 bp repeats are responsible for the inhibition of MIEP activity in such cells (Nelson et al., 1987;Lubon et al., 1989;Kothari et al., 1991).In vivo, monocytes have been identified as an important site of HCMV latency (Taylor-Wiedeman et al., 1991); these cells carry the viral genome in t...

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. INTRODUCTIONAs with all herpesviruses, human cytomegalovirus (HCMV) is able to maintain a life-long latent infection following primary exposure. In the healthy seropositive, latent virus can frequently reactivate however, this usually results in subclinical symptoms (reviewed by Britt, 1998). Life threatening complications arise if the immune system is compromised (i.e. in AIDS and transplant recipient patients) or if infection occurs in utero (reviewed by Britt, 1998). HCMV productively infects a broad array of cell types during viraemia (Sinzger et al., 1995(Sinzger et al., , 1999Sinzger & Jahn, 1996), but in healthy seropositive individuals virus is maintained latently in the myeloid lineage (Mendelson et al., 1996;Minton et al., 1994;Taylor-Wiedeman et al., 1991). It has also been shown, using peripheral blood monocytes (PBMs) (Ibanez et al., 1991;Lathey & Spector, 1991;Soderberg-Naucler et al., 1997;Taylor-Wiedeman et al., 1994) and model cell systems (Gonczol et al., 1984;Weinshenker et al., 1988), that there is a clear correlation between permissiveness of cells for viral immediate-early (IE) gene expression and their state of terminal differentiation. Recently, we showed that in such undifferentiated cells, which are non-permissive for HCMV IE gene expression, repression of the viral major IE promoter (MIEP) is correlated with a closed chromatin conformation and hypoacetylation and hypermethylation of histones around the MIEP (Murphy et al., 2002). In contrast, in differentiated permissive cells, the viral MIEP became associated with hyperacetylated histones consistent with its transcriptional activation (Murphy et al., 2002). Consequently, an analysis of the mechanisms which mediate such differentiation-dependent chromatin remodelling of the vira...

Infection with human cytomegalovirus (HCMV) is known to involve complex interactions between

Infection with human cytomegalovirus (HCMV) modulates the expression of a number of cellular receptors and is known to inhibit expression of the epidermal growth factor receptor (EGFR), a cell surface receptor that can promote cell proliferation through a cascade of intracellular signalling events. We have examined the mechanisms by which HCMV mediates downregulation of EGFR expression and show that virus infection results in the profound upregulation of Wilms' Tumour 1 (WT1) protein, a transcription factor associated with the negative regulation of a number of growth factors and growth factor receptors, including EGFR. Moreover, chromatin immunoprecipitation experiments also show that HCMV infection results in increased binding of WT1 to the EGFR promoter. Finally, we show that depleting the cell of WT1 using small interfering RNA abrogates virus-mediated downregulation of EGFR. Taken together, our observations suggest that HCMV-mediated repression of EGFR expression results from a virusmediated increase in cellular WT1, a known pleiotropic regulator of mitogenesis, apoptosis and differentiation.

EBNA3C is a potent repressor of transcription when bound to DNA as a fusion with the DNA binding domain (DBD) of GAL4. A survey of promoters has revealed that the wild-type, unfused EBNA3C can specifically repress expression from reporter plasmids containing the Epstein-Barr virus Cp latency-associated promoter. Repression of Cp activity required amino acids 207 to 368, which encompasses a region resembling a basic DBD adjacent to a leucine zipper DNA binding motif and a site which binds to the cellular factor CBF1/RBP-J. However, amino acids 207 to 368 are dispensable when the protein is bound to DNA as a fusion with the GAL4 DBD, thus implicating this region in DNA binding. Mutation of the CBF1/RBP-J binding site in EBNA3C abrogated repression, strongly suggesting that CBF1/RBP-J is necessary for targeting the viral protein to Cp. Consistent with this result, mutation of the EBNA2 response element (a CBF1/RBP-J binding site) in Cp also prevented significant repression. In addition, amino acids 346 to 543, which were previously defined as important for the repressor activity of the GAL4-EBNA3C fusion proteins, also appear to be necessary for the repression of Cp. Since repression by these fusions was not observed in all cell types, it seems likely that EBNA3C either depends on a corepressor which may interact with amino acids 346 to 543 or is modified in a cell-specific manner in order to repress. These data are consistent with EBNA3C contributing to the regulation of EBNA expression in latently infected B cells through CBF1/RBP-J and another factor, but this need not directly involve EBNA2. Finally, although it has been reported that EBNA3C can upregulate CD21 in some B cells, we were unable to demonstrate any effect of EBNA3C on reporter plasmids which contain the CD21 promoter.

Human cytomegalovirus (HCMV) is a complex human herpesvirus that is known to productively infect a wide range of cell types. In addition, it has been suggested to contribute to some proliferative disorders, particularly atherosclerosis. Consistent with this, a number of studies have shown that HCMV profoundly affects normal cell cycle control. Specifically, the virus can stimulate early entry into S phase thus ensuring adequate resources for viral DNA replication. Importantly, however, the virus concomitantly inhibits potentially competing cellular DNA synthesis allowing cellular precursors to be used for viral but not cellular DNA replication. The mechanisms by which HCMV perturbs S phase entry involve interactions between the virus and the cellular replication machinery such that formation of competent pre-replication complexes (Pre-RC) at cellular origins of replication is restricted in infected cells.

The expression of Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for the activation and immortalization of human B lymphocytes by EBV. EBNA3C consists of 992 amino acids and includes a potential bZIP motif and regions rich in acidic, proline, and glutamine residues. Thus, EBNA3C resembles several trans regulators of gene expression. It has recently been shown that a fragment of EBNA3C can activate reporter gene expression when fused to the DNA-binding domain of GAL4 (D. Marshall and C. Sample, J. Virol. 69:3624-3630, 1995). Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4. We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression. Repression is dependent on the dose of GAL4-EBNA3C and on the presence of GAL4-binding sites within reporter plasmids. Repression is not restricted to B cells nor is it species or promoter specific. Repression is independent of the location of the GAL4-binding sites relative to the transcription start site. A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues. A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues. We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex.