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...