In this study, we find that HCMV infection stimulates an E2F1-mediated DDR that centers on activation of the ATM kinase early in infection and subsequently coordinates with nuclear viral replication compartments. Moreover, we show that ATM and downstream signaling are required for replication following infection at a low MOI and contributes to HCMV replication at higher doses (). Our results are consistent with ATM contributing to the replication of other viruses (for review, see 
). This conclusion contrasts with what has been previously reported for HCMV infection 
, where it was determined that ATM is not required for the progression of HCMV infection. It is unclear why there is a discrepancy between these studies, but we have confirmed our results using multiple approaches ().
Model showing the relationship between infection, E2F1, ATM activation and HCMV replication.
It appears that ATM activation represents a general response to infection by DNA viruses or viruses that have a DNA stage in the replication strategy, such as retroviruses. The question remains as to why viruses activate ATM and other DDR proteins for replication. Indeed, activation of the host DDR is an obstacle for the replication of at least one DNA virus, adenovirus, which blocks the host DDR during infection 
. One reason for infection-associated ATM activation may be to utilize the consequential stimulation of cellular DNA repair and recombination enzymes 
to benefit viral replication 
. Perhaps, in the case of HCMV, repair and recombination enzymes may aid in circularizing the viral DNA after it has entered the cell and/or facilitate the maturation of nascent viral genomes. A DNA repair complex of DNA ligase IV and XRCC4 circularizes herpes simplex virus genomes early in infection 
. If correct, one would predict that γH2AX, as well as other DDR factors would be bound to virion-delivered HCMV DNA once uncoated in the nucleus. However, others have concluded that parental viral DNA and γH2AX do not co-localize 
. It remains an open question as to whether there is a contribution of the host DDR to very early events in HCMV replication.
HCMV gene expression patterns in infected cells lacking or depleted for ATM may offer clues to the stage(s) in infection that depend on ATM function. IE1 expression is largely unaffected by ATM status. One interpretation of this observation is that ATM does not influence IE events that affect viral replication. Our observation that mature RCs and marker E and L gene products are reduced during infection of AT fibroblasts is consistent with a model wherein ATM influences events associated with DNA replication, presumably by stimulating host (or viral) factors to aid in the repair or recombination of nascent viral DNA. A role for ATM in DNA repair or recombination post replication is also a possibility, although the pattern of viral gene expression argues against this idea. It also cannot be excluded that ATM may have a novel function in phosphorylating/activating an essential host or viral factor not associated with DNA replication, repair, or recombination.
A number of mechanisms have been proposed for how viral infections lead to ATM activation. Upon HIV infection, ATM activation requires the viral integrase and it is proposed that ATM functions in post integration DNA repair 
. For polyomaviruses like SV40, it is thought that the onset of viral DNA replication activates ATM, which then phosphorylates an essential serine residue on large T antigen 
. HPV genome replication appears to switch from theta to rolling circle replication 
, which may activate ATM. Alternately, infection by DNA viruses may cause damage to host chromosomes, which would stimulate a host DDR. In this situation, targets of ATM phosphorylation should also contribute to viral replication.
Both H2AX () and p53 
are substrates of the ATM kinase during HCMV infection and both H2AX () and p53 
contribute to HCMV replication. While the mechanism by which H2AX contributes to HCMV replication is unknown, p53 is found in RCs, binds viral DNA and evidence suggests that p53 influences the expression of viral genes 
. However, the roles of ATM-mediated phosphorylation of H2AX or p53 to productive replication are not known at this point.
Somewhat surprisingly, we find that the initial accumulation of γH2AX following HCMV infection is dependent on ATM whereas ATM is dispensable for γH2AX accumulation once mature viral DNA replication compartments are formed. The kinase(s) responsible for phosphorylating H2AX in the absence of ATM at these later times is unknown but it is possible that another PI3-like kinase, perhaps ATR 
, is responsible for H2AX phosphorylation. DNA PKcs, another PI3-like kinase, is known to phosphorylate H2AX in response to DNA damage signaling, but it has been shown that DNA PKcs does not localize to HCMV RCs 
. However, even though H2AX can be phosphorylated by other kinases later during infection, activated ATM is mostly located in HCMV RCs at these times pi, leaving open the possibility that ATM is influencing activities in these nuclear compartments.
ATM is required for efficient H2AX phosphorylation in MHV68-infected primary marcrophages and ATM is relocalized to sites of viral genome deposition, although a viral kinase also contributes to H2AX phosphorylation 
. ATM is also rapidly relocalized to replication compartments during HSV infection 
. It will be interesting to determine whether ATM is only transiently responsible for host DDR signaling (and viral replication) during infections with herpesviruses.
Deregulation of E2F activity is a hallmark of infections with many DNA viruses that replicate in the nucleus. HCMV infection and expression of its major IE proteins, particular IE1 and IE2, have been shown to inactivate RB family members and induce the expression of E2F regulated genes 
possibly by providing host factors that contribute to virus replication. Our data reveal another consequence of inactivating RB family members and the specific deregulation of E2F1, the activation of a host DDR that facilitates the replication of HCMV.
The mechanism by which E2F1 stimulates host DDR is not known. Inactivation of RB and the subsequent deregulation of E2F1—but not the related family members, E2F2 or E2F3, which also interact with RB—leads to an accumulation of DNA double-strand breaks in human fibroblasts 
. Although it is not clear if HCMV infection causes extensive host DNA damage, infection can result in a DNA double strand break on chromosome 1 
. Whether this single DNA break is sufficient to initiate the observed host DDR is unclear. Alternately, it has been shown that activation of a DDR does not necessarily require DNA lesions. Rather, the physical interaction of DNA repair factors with chromatin can be sufficient to activate the DDR signaling cascade 
. Therefore, host chromosomal changes mediated by disruption of RB/E2F1 complexes or other mechanisms of E2F1 deregulation should also be considered as possible ATM activators during infection.
Most productive infections by DNA viruses result in deregulation of E2F activity through inactivation of RB and RB family members 
. These viruses also activate an ATM-centric DDR, although some viruses, including MHV68, KSHV, and adenovirus, encode factors that can block signals from reaching ATM or its targets 
. The herpesviral proteins responsible for this inhibition are often expressed during latency, which raises the possibility that the host DDR interferes with aspects of latency such as cell survival, proliferation or, perhaps, the maintenance of viral episomes. Most of these viruses have in common infection-associated E2F deregulation, DDR activation, and a contribution of ATM to productive infections. These shared features raise the possibility that E2F1 contributes to the replication of many viruses through its activation of the ATM-associated DDR. It will be interesting to determine how common the E2F1-mediated DDR is to productive viral infections.