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...

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G0-G1 phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.

The influence of caffeine on cytotoxicity and postreplication repair of DNA was examined following exposure of several cell types to physical and chemical agents known to damage DNA. The cell types used in this study were normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), Chinese hamster V79 cells, mouse BALB/c-3T3 cells, and secondary Syrian hamster embryo cells. The DNA damaging agents were ultraviolet light (UV), N-2-acetoxy-fluorenylacetamide (AFAA), nitroquinoline-N-oxide (NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Induction of cytotoxicity in Chinese hamster V79 cells due to ultraviolet light or AFAA exposure was enhanced by caffeine at a concentration of 1.0 mM in the culture medium, but not at 0.2 or 0.05 mM. Caffeine also inhibited postreplication repair in these cells at the same concentrations. In contrast, postreplication repair was not affected by caffeine at concentrations up to 1.0 mM in normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), secondary Syrian hamster embryo cells, and mouse BALB/c-3T3 cells following treatment with ultraviolet light, AFAA, NQO, or MNNG. Cytotoxicity in BALB/c-3T3 cells following exposure to ultraviolet light or AFAA was enhanced in the presence of caffeine at 1.0 or 0.2 mM, although these concentrations of caffeine had no effect on postreplication repair in these cells. The inhibitory effect of caffeine on postreplication repair was found only in Chinese hamster V79 cells among the five cell types used in this study. Both normal and xeroderma pigmentosum human cells repaired mutagen-induced DNA damage equally well in the absence or presence of caffeine at concentrations of 1.0 mM or less.