Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response

Abstract

Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

Figure 1

Steps in the retroviral DNA integration process.

Figure 2

Effect of caffeine on transduction by HIV-1- and ASV-based vectors. (A) HeLa cells were infected with the HIV-1(lacZ) vector and exposed to caffeine for 24 h, as described in Materials and Methods. Five days postinfection, cells were stained with a β-galactosidase assay and blue colonies were counted. (B) HeLa cells were infected with the ASV neo^r vector and exposed to caffeine for 24 h. After removal of caffeine, G418 was added to a final concentration of 1 mg/ml. G418-resistant colonies were counted 6 days postinfection. (C) Micrograph of HIV-1-transduced cells in the presence or absence of 4 mM caffeine. Cells were stained 3 days postinfection (see Materials and Methods). (Magnification: ×200.)

Figure 3

Effect of caffeine on the synthesis of viral DNA, its nuclear import, and the yield of host-viral junction DNA. HeLa cells were infected with the ASV vector and treated with caffeine for 24 h, at which time cells were harvested. (A) Quantitation of viral DNA. Real-time PCR was used to determine the amount of viral DNA relative to mtDNA as described in Materials and Methods. Cells were infected at moi 0.1 and the average of two independent experiments analyzed in duplicate are shown. UN, uninfected cells, HI, cells infected with the heat-inactivated virus; 1:10, cells infected with 1:10 dilution of the virus. (B) Nuclear import of viral DNA determined by formation of LTR–LTR circle junctions. Detection of circle junctions in cells infected at moi 1.0 by PCR is described in Materials and Methods. The expected band is indicated by an arrow. Band intensity at 1 μl of DNA was analyzed by a PhosphorImager and is plotted in D as percentage intensity of circle junctions in the absence of caffeine. (C) Detection of host-viral junction DNA. Covalent joining of viral and cellular DNA after infection at moi 0.01 was evaluated by Alu-PCR as described in Materials and Methods. NP, no Alu primer in the first round of PCR (infection in the absence of caffeine); +, positive control (HeLa cells stably transduced by the ASV vector). Results were analyzed by using a PhosphorImager and are plotted in D as percentage intensity in the absence of caffeine. (D) Comparison of the affects of caffeine on the relative amounts of total, nuclear, and host-viral junction DNA after infection with the ASV vector based on quantitation of data in A–C. Open circles and top curve, total viral DNA; open triangles and middle curve, nuclear viral DNA (circle junctions); filled circles and bottom curve, host-viral DNA junctions. The latter percentages represent an average of two experiments, one of which is shown in C.

Figure 4

Inhibition of ATR function leads to a reduction in the number of retroviral transductants and the yield of host-viral junction DNA. (A) Effect of expression of a dominant-negative ATR gene (ATRkd) on transduction by the HIV-1 vector. GM847/ATRkd cells were treated with doxycycline and infected as described in Materials and Methods. Two days after infection, cells were stained with a β-galactosidase assay and blue cells were counted. Gray columns, cells infected with 10⁻² dilution of the virus (moi <0.01); black columns, cells infected with 10⁻³ dilution of the virus. (B) ATRkd protein detected after treatment of GM847/ATRkd cells with 1 or 5 μg/ml doxycycline for 24 h. Cells were harvested and Western blot analysis was performed with an ATR antibody. (C) Growth of cells expressing ATRkd. GM847/ATRkd cells were plated at a density of 10⁵ cells per 60-mm dish in the presence of doxycycline, which was left on the cells for 48 h and then removed. At indicated intervals, cells were harvested and viable cells were counted. Open circles, cells grown in the absence of doxycycline; crosses, cells treated with 1 μg/ml doxycycline; squares, cells treated with 5 μg/ml doxycycline. (D) Detection of host-viral junction DNA in cells that overexpress ATRkd. GM847/ATRKd cells were treated with doxycycline and infected with the ASV vector. Alu-PCR was performed 24 h postinfection as described in Materials and Methods. NP, no Alu primer in the first round of PCR (cells infected in the absence of doxycycline); UN, no virus (uninfected cells).