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Title: Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle}
Article:
Abstract:
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.
Introduction:
Human cytomegalovirus( HCMV) is a wide-spread human pathogen causing serious disease in immunocompromised patients and neonates[1]. As with all herpesviruses, HCMV exists either in a latent, asymptomatic state or undergoes poductive replication leading to lysis of the host cell. Lytic replication starts with the onset of viral immediate early( IE) gene expression. IE gene products, especially the major IE( MIE) proteins IE1 and IE2, have essential functions in host cell regulation and in activating the subsequent cascade of viral early and late gene expression[2]. In latently infected cells, MIE gene transcription is silenced and consequently viral gene expression is restricted to only very few genomic loci[3],[4],[5],[6]. Reactivation from latency is achieved by mechanisms that trigger desilencing of the MIE promoter/enhancer[7],[8],[9]. Thus, control of MIE gene expression is pivotal to the outcome of infection and, therefore, represents a main focus of HCMV research. In addition, MIE gene expression as the initial step in HCMV replication is considered a prime target for antivirals and an IE2-specific antisense-RNA( fomivirsen) has already proven to be effective in the local treatment of HCMV retinitis[10]. Interestingly, latent infection is not the only situation where HCMV replication is blocked at the level of MIE gene expression. For primary fibroblasts it has been shown that the cell cycle state at the onset of infection determines whether viral gene expression is initiated or not. In G0/G1, IE gene expression starts immediately while in S/G2 phase, transcription of IE1 and IE2 is efficiently suppressed[11],[12]. However, infection of S/G2 fibroblasts does not fully prevent but rather delays the onset of the lytic cycle until cells have completed cell division and reentered the next G1 phase. The physiological relevance of the cell cycle dependent regulation of HCMV is not understood. Furthermore, it is unclear what makes S/G2 cells non-permissive for MIE gene expression and whether the underlying mechanism also plays a role in the establishment of HCMV latency. Here we analyzed the molecular determinants of cell cycle dependent repression of HCMV major IE genes. We found that inhibition of cyclin dependent kinase activity either by checkpoint activation or the chemical inhibitor roscovitine was sufficient to fully restore virus permissiveness in S/G2. Moreover, CDK inhbition was also successful in antagonizing the silencing of lytic gene expression during quiescent, latent-like infection of undifferentiated NTera2( NT2) cells, suggesting a mechanistical link between cell cycle and latency-associated repression of IE gene transcription.
Discussion:
Here we show for the first time that CDK activity negatively controls the onset of HCMV gene expression. This finding was unexpected because HCMV, like many other viruses, was previously shown to be subject of positive regulation by CDKs. Apparently, the effect of CDK activity on HCMV varies depending on the phase of infection and the type of CDK( see figure 12). At a pre-immediate-early stage of infection, an S/G2-specific, probably Cyclin A2-dependent( see below) CDK prevents the initiation of IE gene expression at the level of transcription( this study and our own unpublished data). Once IE expression has been initiated, CDK activity is required for accurate processing and accumulation of viral transcripts[22]. This correlates with recruitment of CDK7 and CDK9 to the sites of viral transcription where they catalyze hyper-phosphorylation of the RNA polymerase II C-terminal domain[23],[24]. At later times CDK activity is needed for proper expression, modification and localization of pUL69, pUL83 and other HCMV proteins and for efficient production of viral progeny[40],[41],[42]. Both CDK2[43] and CDK9[40] but not CDK1[44] are likely to contribute to these late effects. When CDKs are inhibited througout infection, the net effect is an almost complete suppression of HCMV replication[43],[44]. Accordingly, CDKs have been suggested as targets for anti-HCMV therapy[45],[46]. However, our data raise a possible caveat about the use of CDK inhibitors as antiviral drugs because they might favour HCMV induction in non-permissive cell types and during latent or latent-like infections. Several lines of evidence argue that the CDK activity leading to inhibition of MIE gene expression in S/G2 is provided by Cyclin A2-CDK1/2. First, CDK1 and CDK2 are the only CDKs inhibited by both p21( binds CDK1, 2, 4 and 6[47]) and roscovitine( targets CDK1, 2, 5, 7 and 9[48]). Second, Cyclin A2-dependent kinase activity is induced at the G1/S transition and remains high until early prometaphase[49], thus constituting the only Cyclin-CDK activity profile that matches the non-permissive cell cycle window for HCMV. Third, HCMV induces high levels of Cyclin E and B-dependent kinase activity but represses Cyclin A2 in an IE2-dependent manner[11],[50],[51],[52]. This points towards a model whereby HCMV – as soon as MIE gene expression has started – evades the inhibitory influence of Cyclin A2-CDK activity. Also in the case of latent-like infection the Cyclin A2-CDK status appears to perfectly correlate with the ability of cells to support MIE gene expression. Retinoic acid-induced differentiation which prevents the establishment of MIE quiescence in NT2 cells is also known to cause downregulation of Cyclin A2( see also figure 11A), induction of the CDK inhibitor p27 and inhibition of CDK2 activity[37]. Work is ongoing in our laboratory to determine the role of Cyclin A2-dependent kinase activity during lytic infection as well in experimental models of HCMV latency. To understand the mechanism of CDK dependent inhibition of HCMV MIE gene expression it will be essential to know the CDK substrate mediating this control. One possibility is that a viral regulator of MIE gene expression changes its activity, expression or localization depending on the CDK status of the host cell. Examples for such viral sensors of cellular CDK activity are the bovine papillomavirus( BPV) protein E1 and the apoptin protein of chicken anemia virus. BPV-E1, an essential factor for the initiation of viral replication, is inactivated and marked for nuclear export in S/G2 by Cyclin A2-CDK2-dependent phosphorylation[53]. In contrast, apoptin, a tumor selective inducer of apoptosis, requires phosphorylation by Cyclin A2-CDK2 for nuclear localization and cell death induction[54]. In the case of HCMV, the CDK substrate needs to be present at pre-IE times of infection, so the best viral candidate factors would be tegument proteins which are delivered as part of the HCMV virion to the host cell. Importantly, a number of tegument proteins have already been decribed to exert functions prior to MIE gene expression[55] and therefore represent possible targets for such a claimed CDK-dependent mechanism. Candidates include pUS24[56], pUL26[57],[58], pUL28/29[59], pUL35[60],[61], pUL47[62], pUL76[63]. The differentiation-dependent subcellular localization of pp71 that regulates its Daxx-neutralizing function in NT2 and THP-1 cells[38],[39] already provides a proof of principle for the control of HCMV tegument proteins by cellular factors immediately after infection. Moreover, the example of the herpes simplex virus protein VP16 demonstrates that the availability of a tegument protein can be decisive for the cell cycle sensitivity of herpesviral IE gene expression[64]. How might HCMV benefit from a CDK-sensitive mechanism controlling MIE gene expression? In a simple view, it enables the virus to synchronize the onset of its lytic cycle with G0/G1- the cell cycle phase that is considered to be most supportive for virus replication. An alternative scenario is that CDK activity is a downstream constituent of a p53 sensitive switch that can operate between latent and lytic infection. In this case cell cycle dependency of HCMV may be an additional by-product. P53 is a coordinator of cellular responses to different kinds of stress including inflammatory[65] and oncogenic stress[66]. Given that HCMV is frequently found reactivated in inflammatory diseases and cancer[67], the possibility that activated p53 triggers lytic gene expression via CDK inhibition appears an attractive option.
Cyclin-dependent kinases ( CDKs ) control the cell division cycle . Many viruses employ CDK activity to control critical steps of their own replication cycle and to synchronise their replication with the cell cycle dependent availability of vital cellular enzymes and molecular building blocks . Here we show an unexpected antiviral function of CDK activity at a very early stage of human cytomegalovirus ( HCMV ) infection , the onset of immediate early ( IE ) gene expression . HCMV is unique amongst herpesviruses in being unable to initiate IE gene expression during the S/G2 phase of the cell cycle . CDK inhibition by either DNA damage-dependent induction of the cellular CDK inhibitor p21 or by the pharmacological CDK inhibitor roscovitine overcomes this limitation and makes S/G2 cells fully permissive for HCMV . Importantly , in undifferentiated NTera2 ( NT2 ) cells , which normally establish a quiescent , latent-like HCMV infection , CDK inhibition also relieves the block of IE gene expression , suggesting a more general role for CDK activity in the control of this important human pathogen .