ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 206-209
Vol. 34, No. 2
0066-4804/90/020206-04$02.00/0 Copyright © 1990, American Society for Microbiology
5-Azacytidine and 5-Azadeoxycytidine Inhibit Human Immunodeficiency Virus Type 1 Replication In Vitro JACQUES BOUCHARD,1t* MARY CLARE WALKER,2 JEAN-MARIE LECLERC,3 NORMAND LAPOINTE,3 RAYMOND BEAULIEU,4 AND LISE THIBODEAU2 Institut National de la Recherche Scientifique-Sante, Universite du Quebec, Pointe-Claire, Quebec, H9G JR6,1 Immunology and Virology Research Centers, Institut Armand-Frappier, Universite du Quebec, Laval, Quebec, H7N 1B7,2 Pediatric Research Center, H6pital Ste-Justine, Universite de Montreal, Montreal, Quebec, H3T I C5,3 and H6pital Hotel-Dieu, Universite de Montreal, Montreal, Quebec, H2W JT8,4 Canada Received 25 July 1989/Accepted 30 October 1989 Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 ,uM) almost completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-Azacytidine (1 ,iM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 ,uM would result in pronounced cellular cytotoxicity, the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs.
The effective treatment of acquired immunodeficiency syndrome requires the development or discovery of chemotherapeutic agents which ideally would block the replication of the human immunodeficiency virus (HIV) with little or no toxicity to the patient. Since the reverse transcription of HIV RNA to DNA is unique to and crucial for replication of the virus, the search for effective chemotherapeutic agents has concentrated mainly on inhibitors of reverse transcriptase (RT) (9, 10, 13, 14). To date, only the RT inhibitor zidovudine (AZT) is being used in the clinical management of patients with acquired immunodeficiency syndrome (7). Although AZT has produced significant amelioration in the treated patients, its chronic administration does not eliminate the provirus from the infected cells, and many patients have suffered from hematological toxicity (6, 13). A shortcoming of most proposed inhibitor therapies is that the drugs do not act on or eliminate the provirus itself. Consequently, chemotherapeutic agents which affect the integration of the HIV provirus, its stability, and/or its inducibility would be of considerable value in the treatment of acquired immunodeficiency syndrome, either alone or in combination with RT inhibitors or other antiviral therapies. Two nucleoside analogs that we felt might be effective in an HIV provirus destabilization strategy are the 5-azacytosine derivatives 5-azacytidine (5-AZAC) and 5-azadeoxycytidine (5-AZAdC) (2, 11, 12). These analogs are activated through the pyrimidine salvage pathway by the uridinecytidine kinase and the deoxycytidine kinase, respectively, to become nucleoside monophosphate and then nucleoside
triphosphate derivatives (11, 12). These derivatives are effectively incorporated, in competition with their natural counterparts, into RNA (5-AZAC) or DNA (5-AZAdC and 5-AZAC) (3, 11, 12). This incorporation leads to modifications in the normal properties of the nucleic acids. The fraudulent base, 5-azacytosine, is itself unstable and destabilizes the nucleic acid into which it is incorporated (11, 12). Therefore, it is possible that the substitution of a fraudulent 5-azacytosine residue for a cytosine in either the HIV proviral DNA or the viral RNA could render the virus nonviable. The triphosphorylated derivative of both 5AZAC and 5-AZAdC, when incorporated into host genomic DNA in place of normal cytosine bases, causes the hypomethylation of the newly replicated DNA (3, 8, 11). Given that DNA methylation is postulated to be involved in the control of gene expression (4), a change in the HIV DNA methylation pattern induced by the two analogs could change the stability or the transcription rate of the provirus. We report here an evaluation of the capacities of 5-AZAC and 5-AZAdC, at different molar concentrations, to inhibit HIV type 1 (HIV-1) replication in CEM cells, a human CD4-positive T-cell line. MATERIALS AND METHODS Virus strain and cell line. The 1-232 strain of HIV-1 and the human T-lymphocyte cell line CEM, which were the kind gifts of Luc Montagnier, were used throughout the study. CEM cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 jig of gentamicin per ml. Cell viability was determined by the trypan blue dye exclusion technique. The CEM cells were always -95% viable at the beginning of each experiment.
* Corresponding author. t Present address: Pediatric Research Center, Hopital Ste-Justine, Montreal, Quebec, H3T 1C5, Canada.
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Drugs. The drugs 5-AZAC, 5-AZAdC, and AZT were obtained from Sigma Chemical Co. Infection and antiviral treatment. For all experiments, 4 x 105 CEM cells were infected with HIV-1 at a multiplicity of infection of 0.1 50% tissue culture infective dose per cell and cultured in 24-well tissue culture plates at 37°C in a humidified atmosphere containing 5% CO2. RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 pLg of Polybrene per ml, 2 mM L-glutamine, 50 ,ug of gentamicin per ml, and 10-5 M 2-mercaptoethanol was used for the experiments. The multiplicity of infection of 0.1 was chosen after HIV titration on CEM cells, because syncytium formation and other cytopathic effects began to be observed within 2 days after infection and by 4 days, almost 100% of the CEM cells were destroyed. RT assay. The presence of HIV-1 RT in the supernatants of infected, drug-treated, or control CEM cell cultures was assessed by a modification of the procedure of Gallo et al. (5). Viral particles in 1 ml of supernatant were concentrated by ultracentrifugation and suspended in lysing buffer, pH 7.8 (0.1 M NaCl, 0.01 M Tris hydrochloride, 1 mM EDTA, 0.1% Triton X-100). The incorporation of [3H]dTTP into poly(rA): oligo(dT) by the enzyme over 60 min was measured, in triplicate, in an assay volume of 50 ,ul containing 40 mM Tris chloride, 10 mM MgCl2, 50 mM KCI, 2.0 mM dithiothreitol, 0.9 ,ug of poly(rA), 0.1 ,ug of oligo(dT), 5 U of RNaseguard inhibitor (Pharmacia), and 10 ,uCi of [3H]dTTP. Isotope incorporation was assessed by trichloroacetic acid precipitation over a GF/A filter (Whatman, Inc.). Radioactivity (counts per minute [cpm]) was determined by liquid scintillation counting. Supernatants from an uninfected cell control and a non-drug-treated, HIV-1-infected cell control were included in each set of experiments. The results are expressed as the percent decrease in RT activity, calculated as follows: (drug-treated, HIV-infected CEM cell cpm minus drug-treated, uninfected CEM cell control cpm) divided by (non-drug-treated, HIV-infected CEM cell cpm minus non-drug-treated, uninfected CEM cell cpm) multiplied by 100%. Immunofluorescence assay. Untreated and drug-treated HIV-infected CEM cells were fixed onto glass slides with cold acetone, washed with phosphate buffered saline (pH 7.4), and incubated for 1 h at 37°C with a reference human anti-HIV-1 antiserum. At the end of the incubation period, the cells were washed four to five times with phosphatebuffered saline, rinsed with distilled water, and reacted for 1 h at 37°C with the F(ab')2 fragment of goat anti-human immunoglobulin G conjugated to fluorescein isothiocyanate (Organon Teknika). The fluorescein isothiocyanate conjugate was diluted in a 1% rhodamine-phosphate-buffered saline solution. Cells expressing HIV antigens stained green, and uninfected cells stained red. The fluorescence of stained cells was evaluated by an inverted fluorescence microscope. RESULTS Initially, we tested the toxicity of 5-AZAC and 5-AZAdC for CEM cells at achievable pharmacological levels in plasma- 10' to 106 M. No toxicity was observed in terms of cell viability at concentrations of 10' to 10-' M, and the untreated and drug-treated CEM cells were 95 ± 2% (mean ± standard deviation) viable after 72 h of culture. Even after 72 h of exposure to either drug at 106 M, more than 88 ± 2% of the CEM cells treated with 5-AZAdC and 95 ± 2% of the cells treated with 5-AZAC were viable by trypan blue dye
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FIG. 1. Effects of different molar concentrations of 5-AZAC and 5-AZAdC on the growth and viability of CEM cells. (A and C) Growth of CEM cells over 72 h in the presence of no drug (D) or 1 nM (O), 10 nM (A), 1 p.M (A), 0.1 ,uM (0), or 0.5 ,uM (0) 5-AZAdC (A) or 5-AZAC (C). (B and D) Viability by trypan blue dye exclusion of CEM cells over 72 h in the presence of no drug (O) or 1 nM (-), 10 nM (a), 0.1 ,uM (1u), 0.5 p.M (E). or 1.0 ,uM (0) 5-AZAdC (B) or 5-AZAC (D).
exclusion. Cells treated with 1 ,uM 5-AZAC grew at 50% of the rate of untreated control cells. The growth of the 5-AZAC-treated cells was unaffected in comparison with that of untreated CEM cells (Fig. 1). Next, we evaluated the capacities of 10-8 to 10-6 M 5-AZAC and 5-AZAdC, in comparison with AZT at equimolar concentrations, to inhibit the replication of HIV-1 when added at the same time as the virus (Fig. 2). 5-AZAdC (1 ,uM) inhibited HIV-1 replication most effectively; less than 5% of the treated cells reacted with human anti-HIV antisera by indirect immunofluorescence, and supernatant RT activity was almost completely abrogated (>95%), as was syncytium formation. The effect of 5-AZAC was also maximal at 1 ,uM, but only a 60% decrease in RT activity was observed, although only 5% of the cells expressed viral antigens. At 0.5 and 1 ,uM, AZT was slightly, but consistently, less inhibitory than 5-AZAdC and more inhibitory than 5-AZAC. We then examined the effects of adding each drug (1 ,uM) on HIV replication in CEM cells at 2, 6, 23, 30, and 47 h postinfection (Fig. 3). Both 5-azacytosine derivatives inhibited HIV-1 replication when they were added after the virus had been allowed to attach to and penetrate the CEM cells. However, the degree of inhibition was less than that observed when the derivatives and the virus were added to the cells at the same time. RT activity was reduced by almost 60% when 5-AZAdC or 5-AZAC was added 23 h after infection; 80 to 85% of the treated cells reacted with antiHIV antisera, and many syncytia were present. In contrast, AZT inhibited HIV replication equally well whether it was added at the same time as or 23 h after infection with the virus. Next, we wanted to know how long 5-AZAdC needed to remain in contact with HIV-infected CEM cells to be effective. Eleven hours of exposure to 5-AZAdC proved sufficient
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MOLAR CONCNATINS OF DRUGS (M) FIG. 2. Inhibition of HIV-1 replication in CEM cells by different molar concentrations of 5-AZAC ([1), 5-AZAdC (M), and AZT (A). (A) Average percentage of viral antigen expression by each group of drug-treated and untreated HIV-infected CEM cells 3 days after infection as determined by indirect immunofluorescence. A total of 90 to 95% of untreated, infected cells were observed to be fluorescent 3 days after infection. (B) Mean percent decrease in RT activity in supernatants of each of the drug-treated, infected CEM cells compared with activity in supernatants of untreated, infected cells. There was ±2% variation in replicate RT activity determinations. In these experiments, 1 ml of the supernatant of the uninfected, non-drug-treated CEM cells (cell control) incorporated 1,000 cpm of the [3H]dTTP, and the HIV-infected, non-drug-treated CEM cells (virus-cell control) incorporated between 5 x 106 and 10 x 106 cpm of the [3H]dTTP.
to achieve maximum blockage of HIV replication; even the shortest exposure time, 2 h, resulted in a 77% decrease in RT supernatant activity, and only 18% of the cells were fluorescent (Fig. 4).
DISCUSSION As anticipated, 5-AZAC and 5-AZAdC proved effective inhibitors of HIV-1 replication in CEM cells. The maximum inhibition of HIV replication was achieved when 5-AZAC or 5-AZAdC (1 FLM each) was added to CEM cells at the time of infection with HIV. When added to CEM cells postinfection, both analogs inhibited viral replication to a lesser extent than when added at the same time as the virus. 5-AZAdC was always more inhibitory than 5-AZAC was at the same concentration and under the same assay conditions. Prolonged exposure of various cells to 5-AZAC and 5-
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AZAdC has been noted to have a marked effect on cell viability and growth (11), and some of the effects of the drugs on HIV replication in CEM cells could presumably be due to cell growth inhibition. However, in a series of wash-out experiments, we observed that an exposure of 6 h or less to 0.5 ,uM 5-AZAdC produced a substantial inhibition of HIV replication in infected CEM cells (Fig. 3), whereas uninfected CEM cells treated for 6 h neither were less viable nor grew more slowly than uninfected, non-drug-treated CEM cells. The less-pronounced effect on HIV replication with drug concentrations of 1 p.M, when they were introduced some hours after infection with the virus, also provides evidence that cytotoxicity does not contribute substantially to the antiviral effects of 5-AZAC and 5-AZAdC. The fact that 5-azacytosine derivatives must be present early in the replicative cycle of HIV to exert their maximum B
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CYTOSINE ANALOGS INHIBIT HIV-1 REPLICATION
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TIME OF EXPOSUtE AFTER INFEClION (H) FIG. 4. Effects on viral replication of 2, 6, 11, or 23 h of exposure to 5-AZAdC by HIV-1-infected cells. At 2, 6, 11, or 23 h after HIV-1 infection and the addition of 5-AZAdC, quadruplicate cultures of the drug-treated, infected cells were washed three times with RPMI 1640 medium and then suspended and put back in culture in fresh medium supplemented as described in Materials and Methods for the remainder of the 3-day incubation period. The attachment and penetration of HIV-1 into CEM cells at a multiplicity of infection of 0.1 occurs within 1 h. (A) Percent viral antigen expression by immunofluorescence by 5-AZAdC-treated, HIV-1-infected cells at each exposure time. (B) Percent decrease in supernatant RT activity of 5-AZAdC-treated, infected cells at each exposure time, compared with the RT activity in untreated, infected-cell supernatants. One milliliter of supernatant from the uninfected, non-drug-treated CEM cell controls incorporated 1,000 cpm of the [3H]dTTP, and 1 ml of supernatant from each of the four HIV-infected, non-drug-treated CEM cell controls incorporated between S x 106 and 10 x 106 cpm of the [3H]dTTP.
effect is consistent with an antiviral mechanism of action entailing destabilization of the HIV proviral DNA. It is not yet known if methylation of the integrated HIV provirus is involved in maintaining latency. This possibility is supported by the observation that human T-cell lymphotropic virus type I, another human retrovirus, has its proviral DNA sequences normally methylated in nonproductive cells, whereas its long terminal repeat regions are hypomethylated in productive cells (9). This possibility is further supported by the observation that the HIV long terminal repeat enhancer which directs chloramphenicol acetyltransferase gene expression is methylated when it is transfected into Vero or murine cells (1). After treatment with 5-AZAC, the transfected chloramphenicol acetyltransferase-long terminal repeat gene construct is hypomethylated and is expressed, suggesting that methylation of the viral DNA modulated the expression of the HIV provirus (1). Having determined that 5-AZAC and 5-AZAdC do indeed inhibit HIV replication, we are now in the process of testing our hypotheses regarding their mechanism of action in doing so.
ACKNOWLEDGMENTS We thank L. Montagnier for supplying the CEM cells, strain 1-232 of HIV-1, and human anti-HIV antisera and Anne Philippon and Marie-Claire Laverdure for typing the manuscript. This work was supported in part by Research Funds from the Reseau de l'Universite du Quebec and the Cancer Research Society, Inc.
LITERATURE CITED 1. Bednarik, D. P., J. D. Mosca, and N. B. K. Raj. 1987. Methylation as a modulator of expression of human immunodeficiency virus. J. Virol. 61:1253-1257. 2. Bouchard, J., J. M. Leclerc, L. Thibodeau, and M. C. Walker. 1987. 5-AZA-cytosine derivative chemotherapy in AIDS. Ann. Inst. Pasteur Virol. 139:309-318.
3. Bouchard, J., and R. L. Momparler. 1983. Incorporation of 5-AZA-2'-deoxy-5'-triphosphate into DNA: interactions with mammalian DNA polymerase and DNA methylase. Mol. Pharmacol. 24:109-114. 4. Cedar, H. 1988. DNA methylation and gene activity. Cell
53:3-4. 5. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequent detection and isolation of cytopathic retrovirus (HTLV-III) from patients with AIDS. Science 224:500-502. 6. Gill, P. S., M. Rarick, R. K. Brynes, D. Causey, C. Louseiro, and A. M. Levine. 1987. Azidothymidine associated with bone marrow failure in the acquired immunodeficiency syndrome (AIDS). Ann. Intern. Med. 107:502-505. 7. Hirsch, M. S. 1988. Azidothymidine. J. Infect. Dis. 157:427-431. 8. Jones, P. A. 1985. Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation. Pharmacol. Ther. 28:17-27. 9. Kitamura, T., M. Takano, H. Hoshino, K. Shimotohno, M. Shimoyama, M. Miwa, F. Takaku, and T. Sugimura. 1985. Methylation pattern of human T-cell leukemia virus in vivo: pX and LTR regions are hypomethylated in vivo. Int. J. Cancer 35:629-635. 10. Mitsuya, H., and S. Broder. 1987. Strategies for antiviral therapy in AIDS. Nature (London) 325:773-778. 11. Momparler, R. L. 1985. Molecular, cellular and animal pharmacology of 5-AZA-2'-deoxycytidine. Pharmacol. Ther. 30:287299. 12. Vesely, J. 1985. Mode of action and effects of 5-azacytidine and its derivatives in eukaryotic cells. Pharmacol. Ther. 28:227-235. 13. Yarchoan, R., and S. Broder. 1987. Development of antiretroviral therapy for the acquired immunodeficiency syndrome and related disorders. N. Engl. J. Med. 316:557-564. 14. Yarchoan, R., K. J. Weinhold, H. K. Lyerly, E. Gelmann, R. M. Blum, G. N. Shearer, H. Mitsuya, J. M. Collins, C. E. Myers, R. W. Klecker, P. D. Markham, D. T. Durack, S. N. Lehrmann, D. W. Barry, M. A. Fischl, R. C. Gallo, D. P. Bolognesi, and S. Broder. 1986. Administration of 3'-azido-2'deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS and AIDS-related complex. Lancet i:575-580.
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0066-4804/90/020206-04$02.00/0 Copyright © 1990, American Society for Microbiology
5-Azacytidine and 5-Azadeoxycytidine Inhibit Human Immunodeficiency Virus Type 1 Replication In Vitro JACQUES BOUCHARD,1t* MARY CLARE WALKER,2 JEAN-MARIE LECLERC,3 NORMAND LAPOINTE,3 RAYMOND BEAULIEU,4 AND LISE THIBODEAU2 Institut National de la Recherche Scientifique-Sante, Universite du Quebec, Pointe-Claire, Quebec, H9G JR6,1 Immunology and Virology Research Centers, Institut Armand-Frappier, Universite du Quebec, Laval, Quebec, H7N 1B7,2 Pediatric Research Center, H6pital Ste-Justine, Universite de Montreal, Montreal, Quebec, H3T I C5,3 and H6pital Hotel-Dieu, Universite de Montreal, Montreal, Quebec, H2W JT8,4 Canada Received 25 July 1989/Accepted 30 October 1989 Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 ,uM) almost completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-Azacytidine (1 ,iM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 ,uM would result in pronounced cellular cytotoxicity, the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs.
The effective treatment of acquired immunodeficiency syndrome requires the development or discovery of chemotherapeutic agents which ideally would block the replication of the human immunodeficiency virus (HIV) with little or no toxicity to the patient. Since the reverse transcription of HIV RNA to DNA is unique to and crucial for replication of the virus, the search for effective chemotherapeutic agents has concentrated mainly on inhibitors of reverse transcriptase (RT) (9, 10, 13, 14). To date, only the RT inhibitor zidovudine (AZT) is being used in the clinical management of patients with acquired immunodeficiency syndrome (7). Although AZT has produced significant amelioration in the treated patients, its chronic administration does not eliminate the provirus from the infected cells, and many patients have suffered from hematological toxicity (6, 13). A shortcoming of most proposed inhibitor therapies is that the drugs do not act on or eliminate the provirus itself. Consequently, chemotherapeutic agents which affect the integration of the HIV provirus, its stability, and/or its inducibility would be of considerable value in the treatment of acquired immunodeficiency syndrome, either alone or in combination with RT inhibitors or other antiviral therapies. Two nucleoside analogs that we felt might be effective in an HIV provirus destabilization strategy are the 5-azacytosine derivatives 5-azacytidine (5-AZAC) and 5-azadeoxycytidine (5-AZAdC) (2, 11, 12). These analogs are activated through the pyrimidine salvage pathway by the uridinecytidine kinase and the deoxycytidine kinase, respectively, to become nucleoside monophosphate and then nucleoside
triphosphate derivatives (11, 12). These derivatives are effectively incorporated, in competition with their natural counterparts, into RNA (5-AZAC) or DNA (5-AZAdC and 5-AZAC) (3, 11, 12). This incorporation leads to modifications in the normal properties of the nucleic acids. The fraudulent base, 5-azacytosine, is itself unstable and destabilizes the nucleic acid into which it is incorporated (11, 12). Therefore, it is possible that the substitution of a fraudulent 5-azacytosine residue for a cytosine in either the HIV proviral DNA or the viral RNA could render the virus nonviable. The triphosphorylated derivative of both 5AZAC and 5-AZAdC, when incorporated into host genomic DNA in place of normal cytosine bases, causes the hypomethylation of the newly replicated DNA (3, 8, 11). Given that DNA methylation is postulated to be involved in the control of gene expression (4), a change in the HIV DNA methylation pattern induced by the two analogs could change the stability or the transcription rate of the provirus. We report here an evaluation of the capacities of 5-AZAC and 5-AZAdC, at different molar concentrations, to inhibit HIV type 1 (HIV-1) replication in CEM cells, a human CD4-positive T-cell line. MATERIALS AND METHODS Virus strain and cell line. The 1-232 strain of HIV-1 and the human T-lymphocyte cell line CEM, which were the kind gifts of Luc Montagnier, were used throughout the study. CEM cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 jig of gentamicin per ml. Cell viability was determined by the trypan blue dye exclusion technique. The CEM cells were always -95% viable at the beginning of each experiment.
* Corresponding author. t Present address: Pediatric Research Center, Hopital Ste-Justine, Montreal, Quebec, H3T 1C5, Canada.
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Drugs. The drugs 5-AZAC, 5-AZAdC, and AZT were obtained from Sigma Chemical Co. Infection and antiviral treatment. For all experiments, 4 x 105 CEM cells were infected with HIV-1 at a multiplicity of infection of 0.1 50% tissue culture infective dose per cell and cultured in 24-well tissue culture plates at 37°C in a humidified atmosphere containing 5% CO2. RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 pLg of Polybrene per ml, 2 mM L-glutamine, 50 ,ug of gentamicin per ml, and 10-5 M 2-mercaptoethanol was used for the experiments. The multiplicity of infection of 0.1 was chosen after HIV titration on CEM cells, because syncytium formation and other cytopathic effects began to be observed within 2 days after infection and by 4 days, almost 100% of the CEM cells were destroyed. RT assay. The presence of HIV-1 RT in the supernatants of infected, drug-treated, or control CEM cell cultures was assessed by a modification of the procedure of Gallo et al. (5). Viral particles in 1 ml of supernatant were concentrated by ultracentrifugation and suspended in lysing buffer, pH 7.8 (0.1 M NaCl, 0.01 M Tris hydrochloride, 1 mM EDTA, 0.1% Triton X-100). The incorporation of [3H]dTTP into poly(rA): oligo(dT) by the enzyme over 60 min was measured, in triplicate, in an assay volume of 50 ,ul containing 40 mM Tris chloride, 10 mM MgCl2, 50 mM KCI, 2.0 mM dithiothreitol, 0.9 ,ug of poly(rA), 0.1 ,ug of oligo(dT), 5 U of RNaseguard inhibitor (Pharmacia), and 10 ,uCi of [3H]dTTP. Isotope incorporation was assessed by trichloroacetic acid precipitation over a GF/A filter (Whatman, Inc.). Radioactivity (counts per minute [cpm]) was determined by liquid scintillation counting. Supernatants from an uninfected cell control and a non-drug-treated, HIV-1-infected cell control were included in each set of experiments. The results are expressed as the percent decrease in RT activity, calculated as follows: (drug-treated, HIV-infected CEM cell cpm minus drug-treated, uninfected CEM cell control cpm) divided by (non-drug-treated, HIV-infected CEM cell cpm minus non-drug-treated, uninfected CEM cell cpm) multiplied by 100%. Immunofluorescence assay. Untreated and drug-treated HIV-infected CEM cells were fixed onto glass slides with cold acetone, washed with phosphate buffered saline (pH 7.4), and incubated for 1 h at 37°C with a reference human anti-HIV-1 antiserum. At the end of the incubation period, the cells were washed four to five times with phosphatebuffered saline, rinsed with distilled water, and reacted for 1 h at 37°C with the F(ab')2 fragment of goat anti-human immunoglobulin G conjugated to fluorescein isothiocyanate (Organon Teknika). The fluorescein isothiocyanate conjugate was diluted in a 1% rhodamine-phosphate-buffered saline solution. Cells expressing HIV antigens stained green, and uninfected cells stained red. The fluorescence of stained cells was evaluated by an inverted fluorescence microscope. RESULTS Initially, we tested the toxicity of 5-AZAC and 5-AZAdC for CEM cells at achievable pharmacological levels in plasma- 10' to 106 M. No toxicity was observed in terms of cell viability at concentrations of 10' to 10-' M, and the untreated and drug-treated CEM cells were 95 ± 2% (mean ± standard deviation) viable after 72 h of culture. Even after 72 h of exposure to either drug at 106 M, more than 88 ± 2% of the CEM cells treated with 5-AZAdC and 95 ± 2% of the cells treated with 5-AZAC were viable by trypan blue dye
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FIG. 1. Effects of different molar concentrations of 5-AZAC and 5-AZAdC on the growth and viability of CEM cells. (A and C) Growth of CEM cells over 72 h in the presence of no drug (D) or 1 nM (O), 10 nM (A), 1 p.M (A), 0.1 ,uM (0), or 0.5 ,uM (0) 5-AZAdC (A) or 5-AZAC (C). (B and D) Viability by trypan blue dye exclusion of CEM cells over 72 h in the presence of no drug (O) or 1 nM (-), 10 nM (a), 0.1 ,uM (1u), 0.5 p.M (E). or 1.0 ,uM (0) 5-AZAdC (B) or 5-AZAC (D).
exclusion. Cells treated with 1 ,uM 5-AZAC grew at 50% of the rate of untreated control cells. The growth of the 5-AZAC-treated cells was unaffected in comparison with that of untreated CEM cells (Fig. 1). Next, we evaluated the capacities of 10-8 to 10-6 M 5-AZAC and 5-AZAdC, in comparison with AZT at equimolar concentrations, to inhibit the replication of HIV-1 when added at the same time as the virus (Fig. 2). 5-AZAdC (1 ,uM) inhibited HIV-1 replication most effectively; less than 5% of the treated cells reacted with human anti-HIV antisera by indirect immunofluorescence, and supernatant RT activity was almost completely abrogated (>95%), as was syncytium formation. The effect of 5-AZAC was also maximal at 1 ,uM, but only a 60% decrease in RT activity was observed, although only 5% of the cells expressed viral antigens. At 0.5 and 1 ,uM, AZT was slightly, but consistently, less inhibitory than 5-AZAdC and more inhibitory than 5-AZAC. We then examined the effects of adding each drug (1 ,uM) on HIV replication in CEM cells at 2, 6, 23, 30, and 47 h postinfection (Fig. 3). Both 5-azacytosine derivatives inhibited HIV-1 replication when they were added after the virus had been allowed to attach to and penetrate the CEM cells. However, the degree of inhibition was less than that observed when the derivatives and the virus were added to the cells at the same time. RT activity was reduced by almost 60% when 5-AZAdC or 5-AZAC was added 23 h after infection; 80 to 85% of the treated cells reacted with antiHIV antisera, and many syncytia were present. In contrast, AZT inhibited HIV replication equally well whether it was added at the same time as or 23 h after infection with the virus. Next, we wanted to know how long 5-AZAdC needed to remain in contact with HIV-infected CEM cells to be effective. Eleven hours of exposure to 5-AZAdC proved sufficient
208
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to achieve maximum blockage of HIV replication; even the shortest exposure time, 2 h, resulted in a 77% decrease in RT supernatant activity, and only 18% of the cells were fluorescent (Fig. 4).
DISCUSSION As anticipated, 5-AZAC and 5-AZAdC proved effective inhibitors of HIV-1 replication in CEM cells. The maximum inhibition of HIV replication was achieved when 5-AZAC or 5-AZAdC (1 FLM each) was added to CEM cells at the time of infection with HIV. When added to CEM cells postinfection, both analogs inhibited viral replication to a lesser extent than when added at the same time as the virus. 5-AZAdC was always more inhibitory than 5-AZAC was at the same concentration and under the same assay conditions. Prolonged exposure of various cells to 5-AZAC and 5-
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AZAdC has been noted to have a marked effect on cell viability and growth (11), and some of the effects of the drugs on HIV replication in CEM cells could presumably be due to cell growth inhibition. However, in a series of wash-out experiments, we observed that an exposure of 6 h or less to 0.5 ,uM 5-AZAdC produced a substantial inhibition of HIV replication in infected CEM cells (Fig. 3), whereas uninfected CEM cells treated for 6 h neither were less viable nor grew more slowly than uninfected, non-drug-treated CEM cells. The less-pronounced effect on HIV replication with drug concentrations of 1 p.M, when they were introduced some hours after infection with the virus, also provides evidence that cytotoxicity does not contribute substantially to the antiviral effects of 5-AZAC and 5-AZAdC. The fact that 5-azacytosine derivatives must be present early in the replicative cycle of HIV to exert their maximum B
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CYTOSINE ANALOGS INHIBIT HIV-1 REPLICATION
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TIME OF EXPOSUtE AFTER INFEClION (H) FIG. 4. Effects on viral replication of 2, 6, 11, or 23 h of exposure to 5-AZAdC by HIV-1-infected cells. At 2, 6, 11, or 23 h after HIV-1 infection and the addition of 5-AZAdC, quadruplicate cultures of the drug-treated, infected cells were washed three times with RPMI 1640 medium and then suspended and put back in culture in fresh medium supplemented as described in Materials and Methods for the remainder of the 3-day incubation period. The attachment and penetration of HIV-1 into CEM cells at a multiplicity of infection of 0.1 occurs within 1 h. (A) Percent viral antigen expression by immunofluorescence by 5-AZAdC-treated, HIV-1-infected cells at each exposure time. (B) Percent decrease in supernatant RT activity of 5-AZAdC-treated, infected cells at each exposure time, compared with the RT activity in untreated, infected-cell supernatants. One milliliter of supernatant from the uninfected, non-drug-treated CEM cell controls incorporated 1,000 cpm of the [3H]dTTP, and 1 ml of supernatant from each of the four HIV-infected, non-drug-treated CEM cell controls incorporated between S x 106 and 10 x 106 cpm of the [3H]dTTP.
effect is consistent with an antiviral mechanism of action entailing destabilization of the HIV proviral DNA. It is not yet known if methylation of the integrated HIV provirus is involved in maintaining latency. This possibility is supported by the observation that human T-cell lymphotropic virus type I, another human retrovirus, has its proviral DNA sequences normally methylated in nonproductive cells, whereas its long terminal repeat regions are hypomethylated in productive cells (9). This possibility is further supported by the observation that the HIV long terminal repeat enhancer which directs chloramphenicol acetyltransferase gene expression is methylated when it is transfected into Vero or murine cells (1). After treatment with 5-AZAC, the transfected chloramphenicol acetyltransferase-long terminal repeat gene construct is hypomethylated and is expressed, suggesting that methylation of the viral DNA modulated the expression of the HIV provirus (1). Having determined that 5-AZAC and 5-AZAdC do indeed inhibit HIV replication, we are now in the process of testing our hypotheses regarding their mechanism of action in doing so.
ACKNOWLEDGMENTS We thank L. Montagnier for supplying the CEM cells, strain 1-232 of HIV-1, and human anti-HIV antisera and Anne Philippon and Marie-Claire Laverdure for typing the manuscript. This work was supported in part by Research Funds from the Reseau de l'Universite du Quebec and the Cancer Research Society, Inc.
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3. Bouchard, J., and R. L. Momparler. 1983. Incorporation of 5-AZA-2'-deoxy-5'-triphosphate into DNA: interactions with mammalian DNA polymerase and DNA methylase. Mol. Pharmacol. 24:109-114. 4. Cedar, H. 1988. DNA methylation and gene activity. Cell
53:3-4. 5. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequent detection and isolation of cytopathic retrovirus (HTLV-III) from patients with AIDS. Science 224:500-502. 6. Gill, P. S., M. Rarick, R. K. Brynes, D. Causey, C. Louseiro, and A. M. Levine. 1987. Azidothymidine associated with bone marrow failure in the acquired immunodeficiency syndrome (AIDS). Ann. Intern. Med. 107:502-505. 7. Hirsch, M. S. 1988. Azidothymidine. J. Infect. Dis. 157:427-431. 8. Jones, P. A. 1985. Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation. Pharmacol. Ther. 28:17-27. 9. Kitamura, T., M. Takano, H. Hoshino, K. Shimotohno, M. Shimoyama, M. Miwa, F. Takaku, and T. Sugimura. 1985. Methylation pattern of human T-cell leukemia virus in vivo: pX and LTR regions are hypomethylated in vivo. Int. J. Cancer 35:629-635. 10. Mitsuya, H., and S. Broder. 1987. Strategies for antiviral therapy in AIDS. Nature (London) 325:773-778. 11. Momparler, R. L. 1985. Molecular, cellular and animal pharmacology of 5-AZA-2'-deoxycytidine. Pharmacol. Ther. 30:287299. 12. Vesely, J. 1985. Mode of action and effects of 5-azacytidine and its derivatives in eukaryotic cells. Pharmacol. Ther. 28:227-235. 13. Yarchoan, R., and S. Broder. 1987. Development of antiretroviral therapy for the acquired immunodeficiency syndrome and related disorders. N. Engl. J. Med. 316:557-564. 14. Yarchoan, R., K. J. Weinhold, H. K. Lyerly, E. Gelmann, R. M. Blum, G. N. Shearer, H. Mitsuya, J. M. Collins, C. E. Myers, R. W. Klecker, P. D. Markham, D. T. Durack, S. N. Lehrmann, D. W. Barry, M. A. Fischl, R. C. Gallo, D. P. Bolognesi, and S. Broder. 1986. Administration of 3'-azido-2'deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS and AIDS-related complex. Lancet i:575-580.
