Permissiveness for human cytomegalovirus (HCMV) infection is dependent on the state of cellular differentiation and has been linked to repression of the viral major immediate early promoter (MIEP). We have used conditionally permissive cells to analyze differential regulation of the MIEP and possible mechanisms involved in latency. Our data suggest that histone deacetylases (HDACs) are involved in repression of the MIEP in non-permissive cells as inhibition of HDACs induces viral permissiveness and increases MIEP activity. Non-permissive cells contain the class I HDAC, HDAC3; super-expression of HDAC3 in normally permissive cells reduces infection and MIEP activity. We further show that the MIEP associates with acetylated histones in permissive cells, and that in peripheral blood monocytes the MIEP associates with heterochromatin protein 1 (HP1), a chromosomal protein implicated in gene silencing. As monocytes are believed to be a site of viral latency in HCMV carriers and reactivated virus is only observed upon differentiation into macrophages, we propose that chromatin remodeling of the MIEP following cellular differentiation could potentially play a role in reactivation of latent HCMV.

The human cytomegalovirus major immediate-early protein IE86 is pivotal for coordinated regulation of viral gene expression throughout infection. A relatively promiscuous transactivator of viral early and late gene transcription, IE86 also acts during infection to negatively regulate its own promoter via direct binding to a 14-bp palindromic IE86-binding site, the cis repression sequence (crs), located between the major immediateearly promoter (MIEP) TATA box and the start of transcription. Although such autoregulation does not involve changes in the binding of basal transcription factors to the MIEP in vitro, it does appear to involve selective inhibition of RNA polymerase II recruitment. However, how this occurs is unclear. We show that autorepression by IE86 at late times of infection correlates with changes in chromatin structure around the MIEP during the course of infection and that this is likely to result from physical and functional interactions between IE86 and chromatin remodeling enzymes normally associated with transcriptional repression of cellular promoters. Firstly, we show that IE86-mediated autorepression is inhibited by histone deacetylase inhibitors. We also show that IE86 interacts, in vitro and in vivo, with the histone deacetylase HDAC1 and histone methyltransferases G9a and Suvar(3-9)H1 and that coexpression of these chromatin remodeling enzymes with IE86 increases autorepression of the MIEP. Finally, we show that mutation of the crs in the context of the virus abrogates the transcriptionally repressive chromatin phenotype normally found around the MIEP at late times of infection, suggesting that negative autoregulation by IE86 results, at least in part, from IE86-mediated changes in chromatin structure of the viral MIEP.Efficient productive infection of cells with human cytomegalovirus (HCMV) is dependent on a temporal cascade of viral gene expression which relies on the finely tuned, coordinated regulation of specific viral gene products at specific stages of the virus infection cycle. Upon infection of permissive cells, the virus undergoes a regulated cascade of gene expression in three broad phases termed immediate early (IE), early, and late, culminating in the release of infectious virions.The major IE genes are the most abundantly transcribed viral genes at immediate-early times of infection, giving rise to two nuclear phosphoproteins, the 72-kDa (IE72) and 86-kDa (IE86) major IE proteins (59, 62, 69). The expression of IE72 and IE86 is driven by the viral major IE promoter/enhancer (MIEP), and these viral gene products have been implicated in playing a pivotal role in the coordinated regulation of viral gene expression. IE86 acts as a very strong but relatively nonspecific transcriptional activator of viral early and late gene expression; its effect is synergized by the viral IE72 protein.The activation of viral promoters by IE86 is likely to be mediated by direct DNA binding of IE86 to specific DNA-binding sites as well as protein-protein interactions with general transcription f...

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...