DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear gammaH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, gammaH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of gammaH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses.

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family and is recognized as a significant pathogen to certain subgroups of the human population. It has become apparent that HCMV manipulation of the host cell cycle as well as the immune response promotes the replication and propagation of the virus. The ability of HCMV to modulate components of the host immune system and the response to infection most likely contributes to the pathology associated with this virus. This review will address the mechanisms HCMV has adapted to modulate the cell cycle to promote viral replication as well as the different ways it can prevent the "death" of an infected cell.

The proper maintenance of the pathways governing cell growth is critical to ensure cell survival and DNA fidelity. Much of our understanding of how the cell cycle is regulated comes from studies examining the relationship between DNA viruses and the mechanisms of cell proliferation control. There are numerous examples demonstrating that viruses can alter the host cell environment to their advantage. In particular, the small DNA tumor viruses, which include adenovirus, simian-virus 40 (SV-40), and human papillomavirus (HPV), can modulate the host cell cycle to facilitate viral DNA replication. Due to the fact that these viruses infect quiescent, non-cycling cells and lack the necessary enzymes and resources to replicate their DNA (e.g. DNA polymerase), the small DNA tumor viruses must activate the host cell replication machinery in order to expedite viral DNA replication. The capacity of these viruses to perturb normal cell proliferation control is dependent upon their oncogene products, which target p53 and members of the Retinoblastoma (RB) family of proteins and inactivate their respective functions. By targeting these key cell cycle regulatory proteins, the small DNA tumor viruses induce the infected host cells to enter S-phase and activate the components involved with host cell DNA synthesis thereby generating an environment that is conducive to viral DNA replication. In contrast, the larger, nuclear-replicating DNA viruses such as those from the family Herpesviridae, do not share the same stringent requirement as the small DNA viruses to induce the infected host cell to enter S-phase. The herpesviruses encode many of the components to stimulate nucleotide biosynthesis and the necessary factors to facilitate virus DNA replication including a viral DNA polymerase and other accessory factors. Additionally, many herpesviruses encode gene products that arrest the host cell cycle, in most instances, prior to the G1/S transition point. Inducing cells to growth arrest appears to be a prerequisite for the replication of most herpesviruses. However, in addition to encoding factors that inhibit the cell cycle, many herpesviruses encode proteins that can promote cell cycle progression in a manner similar to the small DNA tumor virus oncoproteins. By targeting members of the RB family and p53 protein, the herpesvirus proteins induce S-phase and activate S-phase associated factors that playa role in DNA replication. In this manner, the herpesviruses may promote an environment that is favorable for DNA replication. Consistent with the other herpesviruses, human cytomegalovirus (HCMV)induces human fibroblasts to growth arrest. However, in other cell types, virus infection causes cells to enter S-phase. In addition, HCMV replication requires several cellular factors that are present only during S-phase. Furthermore, HCMV induces the activation of S-phase-associated events as well as the increased expression of numerous S-phase genes following infection. HCMV encodes two immediate early (IE) gene products, IE1-72 and IE2-86, which can interact with members of the RB family of proteins. Additionally, the IE2-86 protein can bind to and inhibit p53 protein function. Given the functional resemblance between the HCMV IE proteins and the oncoproteins of the small DNA tumor viruses, we hypothesized that expression of the HCMV IE proteins could modulate cell cycle control. Specifically, we determined that expression of either IE1-72 or IE2-86 can induce quiescent cells to enter S-phase and delay cell cycle exit following serum withdrawal. Moreover, IE2-86 mediates this effect in the presence or absence of p53, whereas IE1-72 fails to do so in p53-expressing cells. Furthermore, both IE1-72 and IE2-86 induce p53 protein accumulation that is nuclear localized. Because IE1-72 fails to promote S-phase entry in cells expressing p53 and induces p53 protein levels, the mechanism by which IE1-72 alters p53 levels was examined. IE1-72 elevates p53 protein levels by inducing both p19ARF protein and an ATM-dependent phosphorylation of p53 at Ser15. IE1-72 also promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. As consequence of this IE1-72-mediated increase in p53 levels, p21 protein is induced leading to a p21-dependent growth arrest in cells expressing IE1-72. These findings demonstrate that the HCMV IE proteins can alter cell proliferation control and provide further support to the notion that HCMV, through the expression of its IE proteins, induces S-phase and factors associated with S-phase while blocking cell DNA synthesis, to possibly generate an environment that is suitable for viral DNA replication.

It is widely accepted that small DNA tumor viruses, such as adenovirus, simian virus 40 and papillomavirus, push infected cells into S-phase to facilitate the replication of their genome. Until recently, it was believed that the large DNA viruses (i.e. herpesviruses) functioned very differently in this regard by inducing a G(1) arrest in infected cells as part of their replication process. However, studies over the last 6-8 years have uncovered striking parallels (and differences) between the functions of the major immediate early (IE) proteins of at least one herpesvirus, human cytomegalovirus (HCMV) and IE equivalents encoded by small DNA tumor viruses, such as adenovirus. Similarities between the HCMV major IE proteins and adenovirus IE proteins include targeting of members of the RB and p53 families and an ability of these viral factors to induce S-phase in quiescent cells. However, unlike the small DNA tumor virus proteins, individual HCMV IE proteins target different RB family members. HCMV also encodes several other IE gene products as well as virion tegument proteins that act early during infection to prevent an infected cell from replicating its host genome and from undergoing apoptosis. Here, we review the specifics of several HCMV IE proteins, two virion components, and their functions in relation to cell growth control.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G(2)/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.