Cell Cycle Arrest

A surprising observation regarding the kinetics of HCMV replication was that the initiation of the viral gene expression requires that the cells be in G0 or G1 at the time of infection. When cells are infected near or during S phase, many cells are able to pass through S phase and undergo mitosis prior to the synthesis of IE and early gene products (Salvant et al. 1998; Fortunato et al. 2002). The process of cell cycle arrest appears to be important for the early phase of infection, and proteins carried in the virus particle as well as those expressed at immediate early times contribute to this process (see Fig. 2).

Proteins in the Virus Particle

Two virion proteins, pUL69 and pp71 (pUL82), have been shown to modulate cell cycle progression (see the chapter by R. Kalejta, this volume). As components of the virion tegument, they can function as soon as the virus enters the cells. In the case of pUL69, overexpression of this protein stimulates accumulation of cells in G1 phase of the cell cycle (Lu and Shenk 1999). In addition, cells infected with a virus lacking functional pUL69 do not efficiently undergo cell cycle arrest (Hayashi et al. 2000). This mutant does not replicate to wild type levels, but the growth defect may be attributed to other functions of pUL69.

The deletion of pp71 also creates a virus that is severely impaired for growth. The growth defect can be complemented by expression of the protein in trans (Bresnahan and Shenk 2000; Dunn et al. 2003). pp71 has been shown to interact with the cell growth suppressors Rb, p107, and p130 and to target hypophosphor-ylated forms of these pocket proteins for degradation by the proteasome (Kalejta et al. 2003). Consistent with this activity, pp71 expression in uninfected cells accelerates their progression through G1 phase, but does not change the overall doubling

Fig. 2 Modulation of the cell cycle by proteins in the virus particle and immediate early proteins. The tegument proteins pUL69 and pp71 can exert their effects on the cell cycle upon entry of the virus particle into the cell. pUL69 causes accumulation of cells in G1 phase, while pp71 targets the hypophosphorylated forms of the Rb family of proteins (Rb, p107, p130) and the transcrip-tional inhibitor Daxx for proteasome-mediated degradation. This degradation of the Rb proteins along with their viral-mediated hyperphosphorylation lead to the release of the E2F/DP transcription factors, which activate many genes involved in DNA replication and promotes S phase. The IE1 protein blocks the activity of p130/p107, and IE2 interferes with at least some functions of Rb and p53. Both IE proteins can prevent cells from passage through S phase

Fig. 2 Modulation of the cell cycle by proteins in the virus particle and immediate early proteins. The tegument proteins pUL69 and pp71 can exert their effects on the cell cycle upon entry of the virus particle into the cell. pUL69 causes accumulation of cells in G1 phase, while pp71 targets the hypophosphorylated forms of the Rb family of proteins (Rb, p107, p130) and the transcrip-tional inhibitor Daxx for proteasome-mediated degradation. This degradation of the Rb proteins along with their viral-mediated hyperphosphorylation lead to the release of the E2F/DP transcription factors, which activate many genes involved in DNA replication and promotes S phase. The IE1 protein blocks the activity of p130/p107, and IE2 interferes with at least some functions of Rb and p53. Both IE proteins can prevent cells from passage through S phase time (Kalejta and Shenk 2003) . These results suggest that pp71 delivered to cells as part of the incoming virus particles may stimulate the cell cycle at the beginning of the infection before IE gene expression. The finding that pp71 also interacts with ND10-associated transcription repressor Daxx and promotes its proteasome-mediated degradation suggests an additional function for pp71 in initiating viral transcription at ND10 sites (Hofmann et al. 2002; Ishov et al. 2002; Marshall et al. 2002; Cantrell and Bresnahan 2005; Saffert and Kalejta 2006).

Immediate Early Protein 1

HCMV encodes two IE proteins, IE1-72 and IE2-86, that have been shown to interfere with cell cycle progression in heterologous systems in the absence of infection. Transient expression of IE 1-72 in asynchronously cycling cells stimulates their accumulation in the S and G2/M phases of the cell cycle (Castillo et al. 2000). One possible explanation for this result is that it is due to the interaction of IE1-72 with the pocket protein p107 (Margolis et al. 1995; Poma et al. 1996; Woo et al. 1997; Castano et al. 1998; Hansen et al. 2001; Zhang et al. 2003). IE1-72 alleviates p107-mediated repression of E2F-responsive promoters in transient transfection assays and thus may stimulate S-phase entry. Additionally, IE1-72 can reverse the inhibitory effects of p107 on cdk2/cyclin E kinase activity, which may also facilitate the G/S transition. It has been proposed that the formation of an IE1-72/p107 complex mediates these effects (Poma et al. 1996; Johnson et al. 1999; Zhang et al. 2003); however, the finding that IE1-72 can phosphorylate p107, p130, and E2F proteins (Pajovic et al. 1997) raises the possibility that some of the effects of IE1-72 on transcription and the cell cycle result from its reported kinase activity.

Immediate Early Protein 2

Several studies have shown that transient expression of IE2-86 alters cell cycle progression, with a block at the G1/S boundary in a p53+/+ cell or after entry into S phase in a p53 mutant cell (Murphy et al. 2000; Wiebusch and Hagemeier 2001; Noris et al. 2002; Wiebusch et al. 2003; Song and Stinski 2005) (see the chapter by M.F. Stinski and D.T. Petrik, this volume). In transient transfection assays, deletion of aa 451-579 abolished the ability of IE2-86 to induce G1 arrest in transient assays in U373 cells (Wiebusch and Hagemeier 1999). Perhaps the most convincing evidence that IE2-86 plays a role in cell cycle arrest is a recent study showing that a mutation of aa 548 of IE2-86 from Q to R results in a growth-impaired virus that does not inhibit cellular DNA synthesis or the cell cycle (Petrik et al. 2006). The observation that this mutant IE2-86 could still autoregulate the MIE promoter and activate viral early genes provides further evidence that efficient viral replication also requires the inhibition of host cell DNA synthesis.

Early observations showing that IE2-86 interacts with several proteins regulating the cell cycle, including Rb and p53, made it reasonable to link some of its functions to cell cycle arrest (Hagemeier et al. 1994; Sommer et al. 1994; Speir et al. 1994; Bonin and McDougall 1997; Fortunato et al. 1997). p53 levels are stabilized in infected cells but the expression of its target gene p21 is repressed (Muganda et al. 1994; Bresnahan et al. 1996b; Fortunato and Spector 1998; Chen et al. 2001). In transient expression and in vitro systems, IE2-86 interacts with the C-terminus of p53, and the binding of p53 to target promoters is inhibited (Speir et al. 1994; Bonin and McDougall 1997; Hsu et al. 2004). IE2-86 expression also inhibits the acetylation of p53 and of histones in proximity to p53-dependent promoters (Hsu et al. 2004), and thus IE2-86 may regulate expression of p53 target genes by multiple mechanisms. These effects on protein acetylation may result from downregulation of p300/CBP histone acetyl transferase (HAT) activity, which was detected in a complex with p53 and IE2-86 (Hsu et al. 2004). It is possible that the inhibition of p300/CBP HAT activity is p53-promoter-specific, as IE2-86 did not suppress histone acetylation globally. The biological relevance of these experiments must be considered with caution, given that none were performed in the context of the viral infection.

In permissive cells, the early phase of the infection is associated with the stimulation of many genes encoding proteins that are required for host cell DNA synthesis and proliferation (Hirai and Watanabe 1976; Estes and Huang 1977; Isom 1979; Boldogh et al. 1991; Wade et al. 1992; Browne et al. 2001). Many of these genes are regulated by the E2F/DP transcription factors, which are inhibited by complex formation with the Rb family of proteins. A role for IE2-86 has been suggested by work showing that there is an increase in the steady state levels of RNA from several E2F-responsive genes in human fibroblasts infected with an adenovirus expressing IE2-86 (Song and Stinski 2002). The key question, however, is what are the underlying mechanisms for the activation of these growth regulatory genes in the context of the infection?

Cyclin E expression is regulated by E2F, and the potential role of IE2-86 in its accumulation during the infection has been the focus of several studies (Bresnahan et al. 1998; McElroy et al. 2000; Wiebusch and Hagemeier 2001; Wiebusch et al. 2003). The majority of the experiments have used transient expression assays to examine the regulation of the cyclin E promoter driving a reporter gene. In one study, it was shown that IE2-86 could bind to sequences in the cyclin E promoter in vitro and could activate expression of a cyclin E promoter-driven reporter construct (Bresnahan et al. 1998). Work by Song and Stinski demonstrated that expression of IE2-86 induces synthesis of endogenous cyclin E mRNA, reinforcing the notion that IE2-86 expression upregulates cyclin E in infected cells (Song and Stinski 2002). In contrast, McElroy et al. reported that early viral gene expression, not IE2-86 expression, was necessary for accumulation of cyclin E protein (McElroy et al. 2000). These conflicting results suggest that cyclin E accumulation in infected cells is controlled by several pathways, and that the increase in cyclin E might be regulated at both the mRNA and protein level.

Besides E2F transcription factors, other proteins regulate the expression of cyclin genes. The finding that the architectural transcription factor HMGA2 (high mobility group AT-hook 2) regulates the transcription of cyclin A (Tessari et al. 2003) prompted our lab to determine whether HCMV affects the expression of this protein. HMGA proteins are referred to as architectural transcription factors because of their ability to organize the assembly of nucleoprotein structures (enhanceosomes), resulting in enhancement or repression of transcription. In the case of cyclin A, HMGA2 activates its expression through derepression of the promoter. Our studies showed that the transcription of the HMGA2 gene is specifically inhibited at the RNA level during the infection (Shlapobersky et al. 2006). To determine whether repression of HMGA2 was important for the HCMV infection, a recombinant virus expressing HMGA2 driven by the MIE promoter was constructed. High multiplicity infection with the HMGA2-expressing virus induced the synthesis of cyclin A mRNA and protein and inhibited virus replication. Furthermore, a role for IE2-86, but not IE1-72, in HMGA2 repression was suggested by additional experiments with HCMV recombinant viruses defective in either IE1-72 or IE2-86. We found that the IE1-72 deletion mutant virus CR208 (Greaves and Mocarski 1998) inhibits HMGA2 transcription. In contrast, in cells infected with an IE2-86 mutant virus lacking aa 136-290 (referred to as delta SX)

(Sanchez et al. 2002), HMGA2 expression is not significantly reduced, suggesting that IE2 is involved in the regulation of the HMGA2 promoter. Cyclin A transcription is also induced in cells infected with delta SX, although this effect is slightly delayed relative to HMGA2 expression. The mechanism involving the downregulation of HMGA2 RNA expression by IE2-86 has yet to be determined. An interesting possibility is that it is related to IE2 interaction with histone deacetylase (HDAC), as HMGA2 is an example of a gene that requires HDAC activity for its expression (Ferguson et al. 2003). Cyclin D1 expression also seems to require HDAC activity (Hu and Colburn 2005) and cyclin D1 and HMGA2 exhibit a similar pattern of expression in the wt and delta SX infected cells (R. Sanders, M. Shlapobersky, and D.H. Spector, unpublished data). Since the levels of IE2-86 are significantly reduced in cells infected with delta SX, there may be less inhibition of HDAC activity in these cells.

Was this article helpful?

0 0

Post a comment