To understand the effects of HCMV on the host cell cycle, it is necessary to first present a brief overview of the phases of the cell cycle and the major regulatory proteins involved (see Fig. 1). The cell cycle is a finely orchestrated sequence of
Department of Cellular and Molecular Medicine and The Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093-0712, USA [email protected]
T. E. Shenk and M. F. Stinski (eds.), Human Cytomegalovirus. Current Topics in Microbiology and Immunology 325. © Springer-Verlag Berlin Heidelberg 2008
Fig. 1 Effects of HCMV on the cell cycle. The cell cycle is a tightly regulated process through which the cell replicates its DNA and divides into two daughter cells. G0 is the resting state, and cells are stimulated through growth signals to express cyclin D and enter G1 phase. During G1 phase, the anaphase-promoting complex (APC), an E3 ubiquitin ligase, remains active, and proteins that are not needed by the cell until later in the cell cycle are ubiquitinated and targeted for degradation by the proteasome. A prereplication complex (licensing) is established at cellular origins of DNA replication and cyclin E is induced. Replication of the cellular DNA in S phase requires cyclin A. G2 phase follows S phase and marks the transition prior to cell division in M phase. Both cyclin A and cyclin B are required during the G2/M period. The major guardians of the cell cycle are Rb and p53. Infection of cells during G0/G1 phase induces progression through Gj. However, the normal expression of the cyclin-dependent kinases is disrupted, and the cell cycle is blocked before the replication of cellular DNA. HCMV specifically inhibits the expression of cyclin D and cyclin A, but promotes expression of high levels of cyclin E and cyclin B. In infected cells, the activity of the APC is blocked and licensing of the cellular origins of DNA replication is inhibited. p53 is stabilized, but it cannot activate its target promoters, and the inhibitory activity of Rb on the E2F/DP transcription factors is relieved events by which the cell prepares for division (for review see Sherr 1996, 2000). These events are regulated by heterodimeric kinases that consist of a regulatory cyclin subunit and a catalytic subunit, the cyclin dependent kinase (cdk). Multisubunit E3 ubiquitin ligases that target proteins for degradation by the protea-some also regulate cell cycle progression. A resting cell (in G0 phase) can be induced to enter the cell cycle by a number of proliferative signals including growth factors and serum stimulation. Initially, there is activation of the expression of the D-type cyclins, which form kinase complexes with cdk4 or 6. The phosphorylation of substrates by the cyclin D/cdk4 and cyclin D/cdk6 kinases releases the quiescent cell from its resting state and permits entry into the G1 phase of the cell cycle.
The G1 phase is the period during which multiple transcription factors and genes encoding proteins involved in nucleotide metabolism and DNA replication are induced. In G1 phase, there is a commitment to DNA synthesis and cell division if the necessary requirements are met. DNA is replicated completely and only once during a single cell cycle (Fujita 1999; Diffley 2001; Lei and Tye 2001). During Gj phase, prereplication complexes (pre-RC) assemble at the origins of DNA replication. The multisubunit origin recognition complex (ORC) is the first to bind to the DNA and serves as a nucleation point for the recruitment of other factors. Cdc6 and Cdt1 are recruited to the complex and facilitate the loading of the family of six MCM proteins (Maiorano et al. 2000; Nishitani et al. 2000, 2001; Rialland et al. 2002). Cyclin E expression is induced, and the active cdk2/cyclin E kinase promotes the transition into S phase. Cyclin E also facilitates the formation of pre-RC in a kinase-independent fashion through physical interaction with Cdt1 and the MCM proteins (Geng et al. 2003; Ekholm-Reed et al. 2004; Geng et al. 2007).
At the beginning of S phase, cyclin A accumulates and forms an active kinase complex with cdk2. Regulation of cyclin A occurs at both the protein and mRNA levels (Glotzer et al. 1991; Henglein et al. 1994; Desdouets et al. 1995; Schulze et al. 1995; Zwicker et al. 1995; Zwicker and Muller 1997; Bottazzi et al. 2001; Tessari et al. 2003). In S phase, cdk2/cyclin A and cdc7/Dbf4 complexes mediate the firing of DNA origins of replication and promote DNA replication (for review see Nishitani and Lygerou 2002; Machida et al. 2005). At the same time, Cdt1 is released from the replication complex, and its binding to the ORC is prevented by geminin, a protein that accumulates during S, G2, and M phases. This ensures that DNA replication initiates at each origin only once and prevents polyploidy.
Following the complete replication of the parental chromosomes, cells enter G2 and proteins involved in mitosis (M phase) begin to accumulate. Cdk1 and cyclin B play a major role at this point. Cyclin B first associates with cdk1 to form an inactive complex. At the G2/M transition, dephosphorylation of cdk1 by cdc25 phosphatase induces the activation of the kinase, which in turn promotes the onset of mitosis (Millar and Russell 1992). Cdk1/cyclin B and cdk1/cyclin A complexes phosphorylate many substrates that facilitate condensation of the chromosomes, disassembly of the nuclear envelope, and modification of the cell's architecture to ensure that there is an ordered and even segregation of the chromosomes to the daughter cells. Inactivation of the cdk1 complexes occurs during mitosis by degradation of cyclins A and B through the ubiquitin-depend-ent proteolytic pathway involving the anaphase-promoting complex (APC) E3 ubiquitin ligase and the proteasome (Glotzer et al. 1991). The APC also ubiquitinates geminin and targets it for proteasome degradation as the cells exit mitosis, thereby allowing loading of the pre-RCs onto the chromatin of daughter cells during G1 phase (McGarry and Kirschner 1998). This degradation of the cyclins and geminin continues until the onset of S phase (Brandeis and Hunt 1996).
Several checkpoints throughout the cell cycle ensure that progression will halt if there is DNA damage or aberrant spindle formation (for review see Lukas et al. 2004), thus protecting the integrity of the genome. The tumor suppressors p53 and the Rb family of pocket proteins (Rb, p107, and p130) are the best studied of these checkpoint sentinels. The Rb proteins, in their hypophosphorylated forms, bind to the E2F family of transcription factors, which then function as transcrip-tional repressors. Phosphorylation of the Rb proteins in late G1 dissociates these complexes, allowing the E2F factors to activate transcription of multiple genes, many of which encode proteins required for DNA replication (Dyson 1998). p53
coordinates multiple cellular processes through its activity as a transcriptional activator and repressor in response to stress and growth factors (Vousden and Lu 2002; Slee et al. 2004). Phosphorylation of p53 controls its association with MDM2, which targets p53 for degradation by the proteasome (for review see Lavin and Gueven 2006). In response to DNA damage, nutrient deprivation, and other insults to the cell, p53 levels are stabilized. This can lead to the expression of the cdk inhibitor p21 as well as induction of several pro-apoptotic genes.
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