The demonstration of Chk1 activation during SV40 lytic infection implicates components of DNA damage response pathways in the virus-host cell interaction. The results presented here suggest a role for Chk1 in the inhibition of mitosis, although additional Chk1 functions may contribute to viral replication. Previously, activation of Chk1 has been associated with cell cycle arrest resulting from either DNA damage or incomplete DNA replication (10
). It is unclear whether the Chk1 phosphorylation observed in SV40 lytic infection can be categorized as resulting from stalled DNA replication or DNA damage or whether it represents a novel pathway.
When CV-1 cells were infected with the small t deletion mutant dl888, Chk1 was phosphorylated with the same kinetics as in wt infection, demonstrating that Chk1 phosphorylation was not dependent on small t. In addition, infection by dl888 established that the percentage of cells in >G2 was not directly related to the amount of Chk1 phosphorylation. SV40 dl888-infected cells exhibited a reduced rate of progression from G2 to >G2 but no alteration in the phosphorylation of Chk1 relative to that of wt-infected cells. Considering that wild-type levels of Chk1 phosphorylation are obtained without a significant number of cells in >G2, Chk1 phosphorylation appears to be a late S phase or G2 phase event that contributes to the absence of mitosis in both wt and dl888 infections.
Chk1 phosphorylation triggered by stalled replication forks could be a consequence of viral DNA replication and/or cellular DNA replication. It has been reported that phosphorylation of Chk1 in the DNA replication checkpoint response requires the primase activity, but not DNA replication activity, of DNA polymerase alpha (39
). Both viral and cellular origins fulfill this prerequisite for checkpoint signaling, given that the involvement of host cell DNA polymerase alpha primase in SV40 replication is well established (68
). What remain undefined in this case are the checkpoint signaling events that occur subsequent to RNA primer synthesis at the viral or cellular replication forks. The rate of viral DNA replication does not appear to be a determining factor in Chk1 phosphorylation. The rates of viral DNA synthesis in dl
888 or other small t deletion mutant infections are about half of that seen in wt infections (8
), yet we observed no reduction of Chk1 phosphorylation in dl
888 infection. However, SV40 minichromosomes, which may replicate to a copy number of 105
per cell, are found in a variety of forms, including transcriptional complexes and replicative intermediates, and in various states of maturation to virus particles. It may be that this complex environment places stresses on replication fork function that are sufficient to initiate a checkpoint response.
Cellular replication forks may also be the source of checkpoint signaling. Current examples include Chk1 activation in response to inhibitors of DNA synthesis such as hydroxyurea and aphidicolin. Both of these drugs allow synthesis of the RNA primer but inhibit elongation. Activation of Chk1 by UV light may also be a consequence of slowed progress of replication forks. In the case of lytic infection, there is no obvious inhibition of cellular DNA synthesis but the rate of cellular DNA replication may be influenced by the onset of viral DNA synthesis. We have previously shown that increased viral DNA and the late gene product VP1 can be detected at 8 h after release from mimosine, late in S phase (34
). If any replication factors are limiting, the rate of synthesis at cellular DNA replication forks may be adversely affected by the onset of viral DNA replication.
DNA damage is also a stimulus for activation of the Chk1 pathway. The checkpoint response to IR primarily involves the ATM substrates Chk2, p53, and MDM2, whereas activation of Chk1 by ATR appears to play a backup role (2
). During activation of the S phase checkpoint by IR, an initial rapid phase of ATM signaling is followed by an ATM-independent activation of Chk1 that is required to maintain the S phase checkpoint response (78
). In contrast, the checkpoint response to UV and inhibitors of DNA replication depends primarily on ATR-dependent activation of Chk1 (2
). SV40-induced chromosome damage following infection of normal human cells (41
) was described in the early 1960s, soon after the initial reports of SV40-induced cell transformation and tumorigenesis. The increased incidence of chromosome abnormalities within 24 h after SV40 infection of human skin fibroblasts (71
) and Chinese hamster embryo cells (32
) demonstrates that induction of chromosome damage following SV40 infection can occur quickly. Therefore, the phosphorylation of Chk1 observed in SV40-infected CV-1 cells may be in response to damage generated within the first 36 h of infection. Expression of cloned viral genes has established that large T alone is sufficient for the induction of chromosome instability, including chromatid exchanges, chromosome gaps, and breaks and dicentric chromosomes (54
). However, the mechanism of SV40-induced chromosome damage is not well defined. One possibility is that large T may generate damage indirectly by overriding cell cycle checkpoints and disallowing DNA repair. Loss of the p53-dependent G1
phase checkpoint requires the binding of large T to pRb, but the formation of complexes between large T and p53 is not essential (18
). Large T has also been shown to disrupt formation of DNA repair foci containing MRE11 (9
). Override of the G2
phase and spindle checkpoints is found in cells expressing large T and has been detected within 5 to 10 population doublings following initiation of large T expression in human fibroblasts (6
). The alteration of mitotic control within this time period following large T expression suggests that large T is directly responsible for checkpoint override rather than for generating mutations in mitotic regulators that give a selective advantage.
In this study, mitosis and/or ACC were induced in SV40-infected cells by the Chk1 inhibitor UCN-01 and by the ATM/ATR inhibitor caffeine. These inhibitors were previously used to abrogate the G2
arrest in cells with DNA damage induced by genotoxic agents such as IR and adriamycin. The viability of cells following override of G2
arrest by UCN-01 and caffeine treatments is dependent on the cell type and/or the type of genotoxic agent used (5
). In SV40-infected cells, the induction of normal mitosis in caffeine-treated cells was less frequent than in UCN-01-treated cells. Although UCN-01 and caffeine are both known to inhibit Chk1 activity, the difference in their cellular effects may be explained by the additional regulators influenced by each compound. UCN-01, at concentrations of <100 nM, directly inhibits the in vitro protein kinase activity of Chk1, C-TAK1, and several isoforms of protein kinase C but does not inhibit Chk2 (5
). C-TAK1 is a constitutively active cytoplasmic protein kinase that phosphorylates Ser216 of Cdc25C and thus may be responsible for maintaining Ser216 phosphorylation during interphase (46
). Although both Chk1 and C-TAK1 phosphorylate Cdc25C, they also each phosphorylate additional distinct substrates (44
). Downstream effects of UCN-01 have also been described. Wee1 kinase is upregulated by Chk1 in Xenopus
and fission yeast cells (31
) and is inhibited in UCN-01-treated human and murine cells in an indirect manner (55
). Caffeine, as an inhibitor of ATM and ATR, inhibits the downstream activation of both Chk1 and Chk2 (57
). Additional downstream kinases known to be affected by caffeine inhibition of ATM/ATR include Plk1 (67
) and Plk3 (72
). Both of these protein kinases phosphorylate Cdc25C but at different sites. Plk3 phosphorylates Ser216 (47
), the same site phosphorylated by Chk1, Chk2, and C-TAK1. Plk1 phosphorylates Ser198, which is within the nuclear export signal, and may control nuclear localization (66
). Both Plk1 (19
) and Plk3 (48
) are mitotic regulators, and the effect of UCN-01 on their activity has not been reported. Other ATM or ATR substrates, such as NBS1, BRCA1, and the p53 binding protein 53BP1, may also be involved in the response to caffeine (2
). The complexity of the response to caffeine is indicated by the initial phosphorylation of Chk1 in SV40-infected cells exposed to caffeine for 3 h. Initial phosphorylation of Chk1 may be catalyzed by a caffeine-insensitive kinase, or additional factors may influence caffeine inhibition of ATM/ATR. In summary, the greater tendency of UCN-01 to induce normal mitosis while caffeine stimulated ACC may be explained by differences in additional pathways affected by each compound. Understanding the differences in the responses of SV40-infected CV-1 cells to UCN-01 and caffeine will require selective inhibition of Chk1 and other components of the mitotic regulatory pathways.
A related observation was the gradual increase in the normal and abnormal mitotic CC following treatment of infected cells with UCN-01 or caffeine. Unlike uninfected cells, there was not a peak of mitosis at a particular time point, as would be expected in a synchronized cell population. This failure to restore the normal mitotic program may also be explained by the loss of function of additional regulators influenced by each inhibitor, as described above. A second possibility is the involvement of other G2
/M regulatory pathways, such as the p38 stress kinase (1
). DNA damage caused by IR and UV radiation stimulates different members of a group of four p38 kinase isoforms. In response to IR, the p38γ isoform, along with Chk2, is activated in an ATM-dependent manner (69
). In contrast, UV radiation stimulates the kinase activity of the p38α and -β isoforms. These two isoforms of p38 phosphorylate and inactivate Cdc25B phosphatase, a mitotic initiator (4
). A third possibility is that the presence of viral proteins prevents restoration of normal mitotic timing, even when checkpoint controls are overridden.
A number of viruses avoid entry into mitosis either by G2
phase arrest or by continued DNA synthesis, but the purpose of this block in mitotic entry is not known. There are two general categories of speculation: First, the highly complex structural and functional changes in cells during mitosis may temporarily or permanently disrupt a viral replication cycle. In this case, the virus may have evolved mechanisms by which to inhibit the initiation of mitosis. Second, the absence of mitosis may be a strictly cellular response to the stress of viral infection and a mechanism by which to limit proliferation of infected cells. The activation of Chk1 appears to be a novel mechanism for viral inhibition of mitotic initiation. However, viruses that are known to produce a G2
arrest also interact with pathways upstream from MPF. The Vpr protein of human immunodeficiency virus type 1 arrests cells in G2
). Binding of Vpr to PP2A enhances nuclear transport and consequent dephosphorylation of activating residues on Cdc25C. In the absence of active Cdc25C, MPF remains in its phosphorylated, inactive form (23
). Small t of SV40 also binds to PP2A but, in contrast to Vpr, inhibits PP2A phosphatase activity (73
). Overexpression of small t in normal human fibroblasts results in the accumulation of a 4C population with about 30% of the total population arrested in prometaphase. This mitotic arrest appears to result from a failure of either centrosome formation or duplication (15
). However, an influence of small t on PP2A-mediated dephosphorylation of Cdc25C has not been reported. G2
arrest induced by the human parvovirus B19 is dependent on the viral regulatory protein NS-1. The G2
arrest in B19-infected UT7/Epo-S1 cells is characterized by increased protein kinase activity of MPF and cytoplasmic cyclin B. The absence of any indicators of mitosis suggests that nuclear import of MPF is blocked (42
). Reovirus serotype 3 arrest of human, mouse, and canine cells in G2
phase is mediated by the σ1s protein, which is sufficient to cause G2
phase arrest when overexpressed in mouse cells (52
). This G2
arrest is associated with inhibitory Y15 phosphorylation of the Cdc2 subunit of MPF, resulting in deactivation of MPF (51
phase arrests of polyomavirus (33
)- and human cytomegalovirus (25
)-infected cells have also been described.
The increased expression of mitotic markers and stimulation of MPF activity in response to inhibitors of Chk1 activity suggest that SV40 blocks MPF activation by maintaining the phosphorylation of cyclin B-associated Cdc2 at Tyr15. However, additional factors, such as protein subunits that inhibit or activate MPF, may be involved and affected by Chk1. Further characterization of Chk1 and other components of DNA damage response pathways are required to develop a better understanding of the mechanisms underlying altered cell cycle regulation during infection by SV40 and possibly other viruses exhibiting altered G2 phase control.