The long-term cellular response to DNA damage is controlled by the tumor suppressor p53. It results in cellcycle arrest followed by DNA repair and, depending on the degree of damage inflicted, premature senescence or apoptotic cell death. Here we show that in normal diploid fibroblasts the ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C)-Cdh1 becomes prematurely activated in G2 as part of the sustained long-term but not the rapid short-term response to genotoxic stress and results in the degradation of numerous APC/C substrates. Using HCT116 somatic knockout cells we show that mechanistically premature APC/C activation depends on p53 and its transcriptional target p21 that mediates the signal through downregulation of the APC/C inhibitor Emi1. Cdc14B is dispensable in this setting but might function redundantly. Our data suggest an unexpected role for the APC/C in executing a part of the p53-dependent DNA damage response that leads to premature senescence.

Passage through the restriction point late in G 1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restrictionpoint transition of cells before it stops them from replicating their genome. Our results ®t well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

To allow DNA replication only once per cell cycle, origins of replication are reactivated ('licensed') during each G1 phase. Licensing is facilitated by assembly of the pre-replicative complex (pre-RC) at origins that concludes with loading the mini-chromosome maintenance (MCM) complex onto chromatin. Here we show that a virus exploits pre-RC assembly to selectively inhibit cellular DNA replication. Infection of quiescent primary fibroblasts with human cytomegalovirus (HCMV) induces all pre-RC factors. Although this is sufficient to assemble the MCM-loading factors onto chromatin, as it is in serum-stimulated cells, the virus inhibits loading of the MCM complex itself, thereby prematurely abrogating replication licensing. This provides a new level of control in pre-RC assembly and a mechanistic rationale for the unusual HCMV-induced G1 arrest that occurs despite the activation of the cyclin E-dependent transcription programme. Thus, this particularly large virus might thereby secure the supply with essential replication factors but omit competitive cellular DNA replication.

Many viruses antagonize tumor necrosis factor alpha (TNF-␣) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-␣ receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-␣ has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-␣ responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-␣ on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-␣-mediated reactivation of HCMV.

No abstract

Small interfering RNAs (siRNAs) are the mediators of a sequence-specific process of gene silencing called RNA interference (RNAi). Here, we show that synthetic siRNAs against essential gene products of human cytomegalovirus (HCMV) can trigger RNAi in serum-starved, infected primary fibroblasts, as well as in U373 cells, leading to effective inhibition of viral DNA replication. This opens new possibilities for antiviral strategies and for the analysis of viral and cellular genes important to HCMV physiology.RNA interference (RNAi) is a natural mechanism of posttranscriptional gene silencing that is widely conserved in the world of multicellular organisms and is thought to have evolved as a defence strategy against viruses and transposable genetic elements (Hannon, 2002). The molecular mediators of RNAi are double-stranded RNAs (dsRNAs) of 21-23 nucleotides in length that induce the sequencespecific degradation of homologous RNAs (Fire et al., 1998;Hamilton & Baulcombe, 1999). These short interfering RNAs (siRNAs) have rapidly developed into a powerful experimental tool for manipulating gene expression (McManus & Sharp, 2002). Since exogenous siRNA expression allows the targeted knock-down of virtually any gene, even in mammalian systems (Caplen et al., 2001;Elbashir et al., 2001), siRNA methodology has become broadly applied to many areas, not just for the study of gene functions. At least at an experimental level, siRNA approaches have been shown to be effective against a variety of viruses, amongst them prominent viruses such as human immunodeficiency virus 1 (Capodici et al., 2002;Coburn & Cullen, 2002;Jacque et al., 2002;Martinez et al., 2002;Surabhi & Gaynor, 2002), hepatitis virus C (Kapadia et al., 2003;Randall et al., 2003;Wilson et al., 2003), influenza virus (Ge et al., 2003) and poliovirus (Gitlin et al., 2002).To date, no reports have been published describing the use of siRNA technology in human herpesvirus infection. For human cytomegalovirus (HCMV), the prototype of the Betaherpesvirus family and an important pathogen in the immunocompromised host, this may have been due to technical limitations, since cell culture infection systems typically rely on growth-arrested primary fibroblasts, which in turn are difficult to transfect efficiently. We therefore set out to optimize the standard transfection protocol from the Tuschl laboratory (Elbashir et al., 2002) for use in serum-starved primary human foreskin fibroblasts (CRL-2429 from ATCC) employing the transfection reagent Oligofectamine (Invitrogen). Transfections were performed in 12-well plates with cells at 80 % confluence. Three ml 20 mM siRNA and 3 ml Oligofectamine were diluted in 50 ml and 12 ml Optimem (Invitrogen), respectively. After 5 min at room temperature, both solutions were combined. After 20 min, the cell culture medium was aspirated and replaced by the transfection assay, diluted with 400 ml pre-warmed Optimem. At 12 h post-transfection, siRNAcontaining medium was removed and cells were washed once with pre-warmed medium.In order to...

The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets regulators of the cell division cycle for degradation by the 26S proteasome. Discovered as a key regulator of mitosis, the APC/C has more recently been recognized to also play a limiting role in the control of G(0) maintenance, G(1)/S-transition and DNA-replication. Human cytomegalovirus (HCMV) has been shown to interfere with cell cycle regulation at different levels. It can induce an S phase-prone proliferation program in quiescent cells but at the same time this virus directly inhibits competitive cellular DNA replication. Here we show, that human cytomegalovirus (HCMV) inactivates the G(0)/G(1) APC/C rapidly after infection of quiescent fibroblasts, resulting in the untimely stabilization of APC/C substrates. APC/C inactivation is caused by the dissociation of its positive regulator, Cdh1. Surprisingly, this dissociation is independent from known Cdh1 inhibitors, Emi1 and Cyclin A, suggesting that APC/C-Cdh1 inhibition by HCMV is directly caused by a viral protein or an intermediate cellular factor distinct from Emi1 and Cyclin A. Thus, upon infection of quiescent cells HCMV not only activates the E2F-dependent G(1)/S transcription program but also facilitates protein accumulation of APC/C substrates by rapid Cdh1 dissociation.