In this report we show that wortmannin, an irreversible inhibitor of ATM and DNA-PKCS protein kinases, sensitizes a normal murine pre-B lymphocyte cell line to integrase-dependent retroviral killing (Fig. ). The kinetics of such killing are similar to those observed with DNA-PKCS-deficient pre-B lymphocyte lines derived from scid mice. These results are consistent with the interpretation that the viability of infected normal lymphocytes is partially dependent on the activity of DNA-PKCS. We show further that wortmannin can also increase the sensitivity of scid pre-B cells to integrase-dependent retroviral killing, suggesting that an additional wortmannin-sensitive protein(s) contributes to survival of these cells (Fig. ). As a similar increase in sensitivity was observed after treatment of scid cells with ATM antisense oligonucleotides (Fig. ), we propose that ATM can compensate partially for the loss of DNA-PKCS in such cells.
Using a colony assay in which cell survival is dependent on the expression of a stably transduced, virus-encoded reporter (Neor
) gene, we showed previously that the efficiency of such transduction is reduced by 80 to 90% in scid
cells compared to normal murine fibroblasts (12
). Here we describe a similar loss in retrovirus-mediated transduction of human (HeLa) cells that are treated with wortmannin (Fig. ). We observed a dose-dependent reduction in the number of ASV-transduced colonies, with an IC50
value virtually identical to that determined for inhibition of DNA-dependent protein kinase activity in these cells (3.6 versus 4.0 μM). These decreases were observed at concentrations of the drug that had no effect on HeLa cell viability or colony-forming ability. Similar results were obtained with an HIV-1 vector in which transduction was monitored by an independent method, the expression of β-galactosidase activity in individual cells (Fig. ). These results suggest that some function(s) of the cellular protein targets of wortmannin is required to avoid integrase-dependent cell killing and to allow stable retroviral DNA transduction. The simplest interpretation of these data is that the reduced efficiency of stable transduction is a consequence of IN-mediated cell killing. However, other possibilities, such as reduced growth rate or cell cycle arrest of these adherent cells, cannot be excluded.
The colony assay was also used to determine the effect of wortmannin on the efficiency of retroviral transduction with a panel of mutant human and rodent cell lines that lack either ATM or components of the NHEJ pathway (Table ). In the absence of wortmannin, we observed the expected low level of transduction in cells that lacked the NHEJ components, DNA-PKCS or XRCC4. As these cells are null for expression of these two components, this low level of transduction cannot be due to residual NHEJ activity. However, this transduction was reduced even further in the presence of wortmannin. These results are consistent with our observation of increased scid cell killing in the presence of the drug. The results also indicate that the activity of a second wortmannin-sensitive protein, likely ATM, contributes to the residual transduction observed in these cells. This was confirmed by analyses of two A-T cell lines and the demonstration that the observed hypersensitivity of A-T cells was reversed by expression of ATM cDNA (Table ). We also observed that although there was no significant reduction in colony formation in A-T cells in the absence of wortmannin, transduction was reduced by ca. 90 to 95% with only 1 μM wortmannin, and it was virtually abolished with 5 μM wortmannin (Table ). In the absence of infection, the viability of the A-T cell lines was unaffected by these concentrations of drug. As these A-T cells have no ATM kinase, the relevant target in this case is likely DNA-PKCS. Thus, these results suggest that DNA-PK is essential for the survival of stably transduced cells that lack ATM.
Retrovirus-mediated killing of lymphocyte lines is observed as early as 12 h postinfection (12
) (Fig. ). Therefore, an early event in retroviral life cycle or the proteins mediating this event seems to be inducing scid
cell death. However, the integrase-inactivated virus (IN−
) does not kill scid
cells (Fig. ). Because the IN−
virus can perform all early steps of the retroviral life cycle except integration, none of these steps can account for the scid
cell death. Likewise, the expression of viral proteins cannot be responsible for cell killing because the ASV vector is defective for such expression in mammalian cells, and scid
cells are also killed by an HIV-based vector that expresses no viral proteins but only a β-galactosidase reporter (12
). Thus, we conclude that scid
cell killing is dependent on the presence of an active integrase.
Integration into the host cell genome is an essential step in the replication cycle of retroviruses and retrotransposons. In the first two steps of integration, denoted processing and joining, two nucleotides are removed from the 3′ ends of the viral DNA, and these newly created 3′ ends are then joined to staggered phosphates in the complementary strands of host cell DNA (Fig. ) (16
). In the resulting integration intermediate, 5′ ends of the viral DNA are separated by single-strand gaps of four to six nucleotides from the 3′ ends of the flanking host DNA. The processing and joining reactions have been reconstituted in vitro with purified integrase and model DNA substrates. In vivo, repair of the gaps in the host DNA results in the generation of 4- to 6-bp repeats of host DNA flanking each proviral end, and the final covalent joining of the 5′ ends of the viral DNA to the host DNA. The proteins that catalyze this final step in integration have not yet been identified, but host cell repair enzymes are generally assumed to contribute to the reaction.
Our results indicate that the activities of NHEJ pathway or, in their absence, ATM are required for the formation or survival of stable retroviral transductants. As these cellular functions are implicated in DNA damage monitoring and repair, a plausible explanation for our findings is that the integration of viral DNA into the host genome is sensed as DNA damage in infected cells (Fig. ). If NHEJ components are absent or inhibited, apoptotic cell death or growth arrest may occur due to the inability to repair such damage. As predicted from our previous studies (12
), the inhibition of DNA-PKCS
by wortmannin sensitizes normal cells to retrovirus-mediated cell killing. ATM appears to compensate partially for absence of the NHEJ pathway. However, the exact nature of the DNA damage produced by retroviral infection is unknown. In addition to the short gaps in host DNA introduced during IN-mediated joining of viral and host sequences, the viral DNA itself may contain single-strand interruptions. It seems possible that these discontinuities (30
), or double-strand breaks produced when these regions are replicated, are the relevant signals of DNA damage. Other possible signals are changes in host DNA conformation and/or chromatin structure that may occur as a consequence of viral DNA integration. For example, there is evidence that other components of the NHEJ pathway are involved in chromatin silencing (7
). Finally, the lymphocytic cell killing and transduced colony reduction we observed in wortmannin-treated (and repair-deficient cells) could be due to DNA-damaging activity of free integrase rather than to the integration reaction per se. The latter interpretation seems unlikely, as integrase presumably remains associated with the viral DNA until host target DNA is encountered. It is also inconsistent with the results of our computational analyses of retrovirus-induced scid
cell death (R. Daniel, S. Litwin, R. A. Katz, and A. M. Skalka, unpublished data). However, further studies will be required to address this issue.
The exact manner in which the NHEJ pathway mediates repair of DNA damage is still unknown. A direct role has been proposed, in which the DNA-binding Ku heterodimer subunits attach the DNA-PK complex to the site of damage and allow the recruitment of other necessary components (40
). The protein kinase activity of DNA-PK may be needed for signaling to other proteins or for modification of those recruited to site of damage. In V(D)J recombination, DNA-PK is also thought to interact with the RAG proteins to complete the joining of immunoglobulin coding strands (6
). As the mechanism of V(D)J recombination and retroviral integration seem to be related evolutionarily (1
), it is possible that DNA-PK also interacts with the viral integrase or other viral proteins during integration.
The hypersensitivity of A-T cells to DNA damage is due to a dual defect in DNA repair and checkpoint control (24
). Either one or both of these activities could be relevant to the establishment of a stably integrated provirus. The ATM kinase is required for phosphorylation and activation of p53 and Chk2 proteins, which are implicated in cell cycle arrest and apoptosis (4
). A-T cells are unable to arrest at the G1
/S and G2
/M checkpoints in response to DNA damage, and they also exhibit radiation-resistant DNA synthesis. It is possible that, in the absence of DNA-PK, ATM-mediated growth arrest allows cells to repair the potentially lethal damage introduced by retroviral DNA integration via an alternative, DNA-PK-independent pathway(s). It is also possible that ATM participates directly in the repair of such DNA damage (11
The finding that NHEJ and, in its absence, ATM are required for stable retrovirus-mediated transduction lends further credence to our proposal that the integration intermediate is sensed as DNA damage by the cell (12
). This type of “damage” can be titrated, and its molecular aspects can be studied using the viral sequences as a probe. Further experiments with this system should help us to determine which activities of DNA-PK and ATM are critical and to identify other cellular proteins that may play a role in integration damage repair. An understanding of the mechanisms by which cellular repair proteins contribute to this process could have a practical application. We show here that a drug which blocks cellular repair pathways can inhibit stable transduction by HIV-1, most likely because infected cells cannot survive IN-mediated DNA damage. These results suggest a strategy for antiretroviral (AIDS) therapy that targets cellular proteins rather than viral proteins. Such a strategy would have the advantage of minimizing the potential of selecting for viral escape mutants.