We reported recently that retroviral transduction can be blocked by caffeine, an inhibitor of host cell DNA repair. We have also shown that efficient retroviral transduction requires the cellular ATR protein, which is a known caffeine target. However, only cycling cells were used in experiments described in the initial work (13
). Because nondividing cells, such as macrophages, are also main targets of HIV-1 infection and caffeine is presumed to exert its effect on DNA repair primarily by regulation of cell cycle checkpoints, it could be expected that caffeine will affect retroviral transduction only in cycling cells. The studies described here show that caffeine also inhibits HIV-1 transduction of nondividing cells.
We performed our initial experiments with nocodazole-arrested 293T cells in M phase and observed that caffeine inhibits the HIV-1 transduction in nondividing 293T cells with the same efficiency as in dividing 293T cells. It has been reported that in nondividing cells, unintegrated HIV-1-based vector DNA may be expressed as efficiently as integrated DNA (43
). Therefore, we also infected the growth-arrested cells with a control vector carrying an inactivating D64V substitution in HIV-1 integrase (32
). We observed that the transduction efficiency of this vector is about 10-fold lower than that of the vector with a wild-type integrase gene. Therefore, we conclude that the majority of the observed expression is from integrated proviral DNA.
To determine if caffeine inhibits HIV-1 transduction of cells arrested in G1
phase, we examined contact-inhibited MEFs. As with the nocodazole-arrested 293T cells, we observed that caffeine also reduces transduction of the contact-inhibited MEFs. The majority of the reporter gene expression in these cells was again derived from integrated proviral DNA, as described previously (12
). Caffeine treatment also inhibited HIV-1 transduction of terminally differentiated, postmitotic neurons and macrophages.
Caffeine inhibits checkpoint activation in response to DNA damage, a process that is known to be regulated by the ATM and ATR kinases (1
). Caffeine was found to inhibit catalytic activities of these kinases in vitro, at concentrations that are required to induce radiosensitization in vivo (21
). In vivo, caffeine reduces ATM-mediated Chk2/Cds1 activation and phosphorylation (55
). Therefore, it has been assumed that caffeine exerts its effects by direct inhibition of ATM and ATR kinases, and this drug has been used widely to study the function of ATM and ATR in cultured cells (10
). It was reported very recently that the phosphorylation of some ATM and ATR substrates in cultured cells is not inhibited by caffeine (8
). However, subsequent published results indicate that caffeine does inhibit the ATR and ATM kinases in vivo (5
). As we had shown that ATM-deficient cells are transduced at a normal efficiency (13
), it seemed likely that the observed effect of caffeine on HIV-1 infection is mediated by its inhibition of the ATR kinase.
We examined retroviral transduction of nacodazole-arrested cells that express the dominant-negative ATRkd (6
). Our results demonstrated that HIV-1 transduction of nondividing cells is reduced upon expression of ATRkd in a manner similar to that observed with dividing cells.
As it has been reported that the Vpr protein may trigger the cellular ATR-dependent DNA damage response, we also included an HIV-1-based vector lacking the Vpr gene in our experiments (41
). However, transduction efficiency of this vector was as sensitive to the expression of the ATRkd transdominant mutant as that of the Vpr-carrying HIV-1-based vector (Fig. ). These data suggest that ATR may play at least a dual role in the HIV-1 life cycle, affecting both Vpr-induced growth arrest and retroviral DNA integration.
Finally, to determine if the effect of caffeine on HIV-1 transduction is due to inhibition of the ATR pathway, we treated cells expressing the dominant-negative ATRkd protein with caffeine. We observed that the residual HIV-1 transduction of ATRkd-expressing cells is less sensitive to caffeine treatment than transduction of ATR-proficient cells. These results suggest that the caffeine effect on HIV-1 transduction is at least partly mediated by inhibition of the ATR pathway.
We proposed previously that one possible explanation for the effects of caffeine and ATRkd expression on retroviral transduction is that the retroviral DNA integration intermediate elicits a DNA damage response, which in turn leads to a transient cell cycle arrest that allows time for cellular DNA repair enzymes to complete the required postintegration repair reactions (13
). Our finding that HIV-1 transduction of nondividing cells is also reduced by caffeine and expression of ATRkd argues against a requirement for checkpoint activation. Rather, the studies reported here support the alternative explanation, namely that ATR is directly involved in postintegration repair at sites of retroviral DNA integration, through either recruitment or modification of the necessary repair proteins.