It is becoming increasingly clear that DNA damage response signaling plays an important role in the replication of many viruses. This study represents the first report of a virus stimulating the DNA damage response in invertebrate cells. Our results demonstrate that replication of the baculovirus AcMNPV stimulates signaling through DNA damage response pathways, similar to what has been reported for several DNA viruses that infect vertebrates. Stimulation of the DNA damage response is dependent on initiation of viral DNA replication; however, efficient levels of viral DNA replication in turn depend on the DNA damage response (). It is likely that intermediates formed during viral DNA replication (33
) are interpreted by the cell as damaged DNA and that this is the trigger for the DNA damage response, although at this time we cannot rule out the possibility that a late gene product is responsible. One or more unknown cellular factors activated by the DNA damage response are presumably utilized by the virus to achieve full levels of DNA replication and, thus, late and very late gene expression, which depends on DNA replication. Although the identities of these cellular factors remain to be determined, they may include enzymes involved in DNA repair or recombination.
Fig. 10. Relationship between AcMNPV DNA replication, the DNA damage response, and apoptosis. Solid arrows indicate known relationships, while dashed arrows indicate hypothesized relationships. The initiation of AcMNPV DNA replication stimulates the DNA damage (more ...)
Using caffeine to inhibit DNA damage response signaling caused decreased viral DNA replication and late gene expression and also reduced virus-induced apoptosis. A similar dependence on DNA damage response signaling for optimal virus replication has been reported for several mammalian viruses, including the herpesviruses human cytomegalovirus (HCMV) (12
) and herpes simplex virus type 1 (HSV-1) (24
). Both of these herpesviruses stimulate the DNA damage response via immediate early gene expression (12
), which differs from our results with AcMNPV, where stimulation of the DNA damage response appears to be dependent on viral DNA replication. However, with both HCMV and HSV-1, abrogation of the DNA damage response inhibits viral DNA replication, similar to what we have observed with AcMNPV. In the case of both HSV-1 and HCMV, inhibition of the DNA damage response reduces the formation of mature viral replication centers, perhaps by inhibiting localization of DNA repair enzymes that are necessary for viral replication to the viral centers (7
). It has been noted that DNA replication of baculovirus is similar in many ways to that of herpesviruses, as both appear to be dependent on recombination (45
), and baculoviruses also form nuclear replication foci (37
). Therefore, it will be interesting to determine whether a similar effect on formation of replication centers is observed in AcMNPV-infected cells that are treated with inhibitors of the DNA damage response.
The ability of caffeine to inhibit virus-induced apoptosis is probably due to the fact that caffeine inhibited viral DNA replication of the vAcP35KO-PG mutant, which is the main trigger for virus-induced apoptosis (49
). On the basis of these data, we cannot determine whether the DNA damage response is also involved in apoptosis downstream in the pathway, after induction by viral DNA replication (). One of the main mechanisms by which the DNA damage response triggers apoptosis is through activation of P53, but silencing Sfp53
expression had no effect on apoptosis stimulated by either virus infection or treatment with UV or CPT. In Drosophila
, DmP53 upregulates expression of the proapoptotic gene reaper
after DNA damage (5
). The Reaper protein functions as an inhibitor of apoptosis (IAP) antagonist and can trigger degradation of the protective IAP protein DIAP1 (54
). S. frugiperda
cells also express an IAP protein, SfIAP, that is required for cell viability (35
). It has been shown that AcMNPV infection causes a decline in the levels of SfIAP protein (57
). The decrease in SfIAP is also triggered by viral DNA replication and is partially due to proteosome-mediated degradation (57
), but exactly how viral DNA replication triggers SfIAP degradation is not clear. Because of their interdependent nature, it is difficult to tease apart the processes of baculovirus DNA replication, the DNA damage response, shutoff of host gene expression, and apoptosis (). Thus, the exact role of the DNA damage response in AcMNPV-induced apoptosis is still unclear.
expression had no discernible effect on virus replication. This was not unexpected, since many viruses encode proteins that inactivate P53. P53 is only one of the downstream targets of ATM, and it is likely that other arms of the DNA damage response are responsible for boosting virus replication. However, although P53-independent apoptosis can occur in response to DNA damage (31
), it was somewhat surprising that silencing Sfp53
did not affect apoptosis stimulated by infection or by UV or CPT treatment. In fact, caspase activity was consistently higher in cells where Sfp53
was silenced before treatment. This higher caspase activity could potentially be explained by the ability of SfP53 to stimulate expression of one or more antiapoptotic genes. Similar results have been reported in mammalian cells lacking p53
and are thought to be due to the ability of P53 to stimulate expression of proteins involved in cell cycle arrest and DNA repair, such as the cyclin-dependent kinase inhibitor p21 (13
), although p21 does not appear to be a transcriptional target of P53 in Drosophila
). Regardless, the ability of apoptosis to occur normally, even though levels of SfP53 were drastically reduced by RNAi, suggests that SfP53 is not required for apoptosis stimulated by infection or the DNA-damaging agent UV or CPT in Sf9 cells. It is possible that SfP53 is normally involved in apoptosis triggered by these stimuli, but when SfP53 levels are reduced, other pathways are sufficient for apoptosis. It is also possible that the Sf9 cell line may have undergone genetic alterations that make it less dependent on SfP53 for apoptosis than normal for S. frugiperda
cells. While we cannot rule out the possibility that silencing of Sfp53
did not reduce SfP53 levels enough to affect its function, we consider this unlikely since the kinetics of apoptosis were unaffected by the drastically reduced SfP53 levels (data not shown).
At this time, it is not clear whether the AcMNPV protein Ac92, which binds to human P53 (44
), plays any role in regulating the function of SfP53 during AcMNPV infection. Although Ac92 was shown to enhance apoptosis stimulated by overexpression of human P53, this was done using a protein overexpression approach, the results of which can be difficult to interpret. Infection with AcMNPV stabilizes P53, as does infection with simian virus 40 (SV40). Interestingly, interaction with SV40 large T antigen stabilizes P53 but inhibits P53 function (2
). Although we have observed that Ac92 interacts with SfP53 (unpublished results), the question of whether Ac92 regulates SfP53 function needs to be further investigated.
Our results indicate that the increased abundance of SfP53 that is observed after triggering of the DNA damage response in Sf9 cells is not due to increased Sfp53
transcript levels. Instead, the accumulation of SfP53 is likely due to increased protein stability, as it is in mammalian cells, although this remains to be directly demonstrated. Although DmP53 has been reported to not increase in abundance following irradiation of embryos (6
), an E2 ubiquitin-conjugating enzyme, dRad6, has been reported to affect the stability of DmP53 in Drosophila
cell lines (8
). The difference between our results and those reported in Drosophila
may be due to using embryos versus established cell lines or to a species-specific difference.
In conclusion, we have shown that AcMNPV replication stimulates DNA damage response signaling in Sf9 cells, as demonstrated by overaccumulation of SfP53, H2AX phosphorylation, and the ability of inhibitors of ATM and ATR to affect these processes. In turn, optimal replication of AcMNPV depends on yet-to-be-determined factors stimulated by the DNA damage response. Although induction of apoptosis by AcMNPV depends on viral DNA replication, the downstream effector SfP53 is not required for AcMNPV-induced apoptosis in Sf9 cells. Further investigation will continue to unravel the role of the DNA damage response in AcMNPV replication and viral stimulation of apoptosis.