- Email: [email protected]
Mutagenic outcome of combined antiviral drug treatment during human immunodeficiency virus type 1 replication
Available online at www.sciencedirect.com R
Virology 307 (2003) 116 –121
www.elsevier.com/locate/yviro
Mutagenic outcome of combined antiviral drug treatment during human immunodeficiency virus type 1 replication Louis M. Mansky* Department of Molecular Virology, Immunology, and Medical Genetics, Center for Retrovirus Research, and Comprehensive Cancer Center, Ohio State University Medical Center, Columbus, OH 43210, USA Received 16 August 2002; returned to author for revision 2 October 2002; accepted 14 October 2002
Abstract The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy (ART) is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. To assess the impact of combination therapy on HIV-1 mutagenesis, the mutagenic outcome of combined drug treatment was determined with several different RT drug combinations. Significant increases in HIV-1 mutant frequencies were observed with combinations of nucleoside RT inhibitors as well as in combinations where nucleoside inhibitors were used along with hydroxyurea, a drug known to deplete nucleotide pools in cells. This indicates that combinations of RT drugs can act together to further increase HIV-1 mutant frequencies, which could have important implications for virus population dynamics and could compromise drug therapy regimens. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Nucleoside; Polymerase; Evolution; Drug; Mutagenesis
Introduction The treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals with antiretroviral drugs including reverse transcriptase (RT) and protease inhibitors in a combination therapy (called potent antiretroviral therapy, or ART) has significantly reduced the rate of HIV and AIDS-related morbidity and mortality (Richman, 2001). However, a problem with these therapies is that they can be suboptimal, due in some cases to a lack of patient compliance to drug administration, and allow for the selection of drug-resistant viruses (Bangsberg et al., 2000). Antiviral drugs have been shown to increase virus mutant frequencies of retroviruses and RNA viruses (Mansky and Bernard, 2000; Mansky and Cunningham, 2000). Since the in vivo mutation rate for HIV-1 has been previously determined to be very high (i.e., 4 ⫻ 10⫺5 mutations per target bp per
* Corresponding author. Department of Molecular Virology, Immunology, and Medical Genetics, 2078 Graves Hall, 333 West 10th Ave., Columbus, OH 43210. Fax: ⫹1-614-292-9805. E-mail address: [email protected] (L.M. Mansky).
replication cycle) (Mansky, 1996; Mansky and Temin, 1995), the increased virus mutant frequency in the presence of the drugs could lead to a rapid rate of selection and fixation of mutations that confer drug resistance (Richman et al., 1994; Schuurman et al., 1995). These drug-resistant viruses can readily reside in latently infected cells, which further complicates subsequent drug treatment regimens during the life of the infected individual. When drug-resistance mutations accumulate, drug susceptibility diminishes and reduces the potency of the components of ART. The continued replication in the presence of drug will select for even greater levels of resistance and typically leads to crossresistance to drugs of the same class. Transmission of HIV-1 with reduced susceptibility to antiretroviral drugs may further compromise the efficacy of drug therapy (Garcia-Lerma et al., 2001). In this study, the mutagenic outcome of various drug combinations on HIV-1 replication was analyzed. It was found that virus replication in the presence of nucleoside reverse transcriptase inhibitors (NRTIs) combinations led to an additive increase in virus mutant frequencies. Virus replication in the presence of drug combinations consisting of an NRTI and hydroxyurea,
0042-6822/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0042-6822(02)00069-7
L.M. Mansky / Virology 307 (2003) 116 –121
117
Fig. 1. Assay system used to analyze virus mutant frequencies in vivo during one round of HIV-1 replication using the lacZ gene as a mutation target. A. HIV-1 vector used to measure virus mutant frequencies. The proviral DNA form of the vector is shown. The large black rectangular boxes are the long terminal repeats. The small gray box is the SV40 promoter. The lacZ gene, internal ribosomal entry site (IRES) sequence, and the neo gene are indicated. B. Single-cycle replication assay for mutant frequencies. HeLa cell clones with single integrated vector proviruses were transiently transfected with helper plasmids and the produced virus was used to infect fresh HeLa cells that were treated with drug postinfection (see materials and methods). G418-resistant cells resulting from virus infection of fresh HeLa cells were selected and cells were then stained with X-gal. The ratio of white plus light-blue stained colonies to total colonies was used to determine the forward mutant frequency.
a drug known to deplete dNTP pools in cells, also increased virus mutant frequencies. This indicates that combination drug therapy increases HIV-1 mutant frequencies, which could have important implications for long-term administration of anti-HIV chemotherapy.
Results Dose dependent effect of dd1 on HIV-1 mutant frequencies Various concentrations of ddI, ranging from 0.1 M to 0.8 M, were tested for their effects on virus mutant frequencies using a postinfection treatement strategy (see materials and methods) (Fig. 1). There was a corresponding increase in virus mutant frequency observed (Fig. 2). This indicates that ddI influences the virus mutant frequency in a dose-dependent manner. A dose-dependent relationship between increased drug concentration and increased virus mutant frequency has been previously observed for AZT and 3TC (Mansky and Bernard, 2000), as well as for hydroxyurea (Mansky et al., 2002). The maximum increase in virus mutant frequency in the presence of ddI was 6-fold higher
than the virus mutant frequency during replication in the absence of the drug (P ⬍ 0.0001). Additive effect of NRTI combinations on HIV-1 mutant frequencies Various combinations of NRTIs were tested for their ability to interact together to alter HIV-1 mutant frequencies during virus replication in a manner that was significantly different than observed with individual drugs alone. The drug combinations tested included AZT and 3TC, AZT and ddI, and finally 3TC and ddI. The rationale for testing these combinations was that at least two of these NRTIs are typically used in ART. Control experiments were done with each drug individually to assess the potential interplay between drug combinations in altering virus mutant frequencies. Since each drug individually increased the odds of virus mutants (Fig. 3), it was hypothesized that combined drug treatments would further increase these odds and result in an increase of virus mutant frequencies. Several different outcomes could be envisioned. The drugs could act in an additive manner, a multiplicative manner, or a synergistic manner. In each of the NRTI combinations tested, an additive increase in virus mutant frequencies was observed (Fig.
118
L.M. Mansky / Virology 307 (2003) 116 –121
Fig. 2. Dose-dependent effect of ddI on HIV-1 mutant frequencies. Various concentrations of ddI were analyzed for their influence on HIV-1 replication in one round of replication. Mutant frequencies were determined as described in Fig. 1. The average mutant frequencies (0.15 mutant/cycle at 0 M ddI) were determined from 3 replicate experiments ⫾ SD.
3). In particular, virus replication in the presence of AZT or ddI alone led to 4-fold and 2.8-fold increases in the odds of recovering virus mutants, respectively. However, during HIV-1 replication in the presence of both AZT and ddI, the virus mutant frequency was 6.5-fold higher than observed in
the absence of drug. This 6.5-fold increase in mutant frequency best fits an additive model (i.e., 4 ⫹ 2.8 ⫽ 6.8) versus that of a multiplicative model (i.e., 4 ⫻ 2.8 ⫽ 11.2) or a synergistic model (significantly higher than 11.2-fold). As with the AZT/ddI combination, the combination of 3TC
Fig. 3. Additive effect of nucleoside reverse transcriptase inhibitor (NRTI) combinations on HIV-1 mutant frequencies. Various combinations of NRTIs were analyzed for their influence on HIV-1 replication in one round of replication. The average mutant frequencies (0.14 mutant/cycle for no drug) were determined from 3 replicate experiments ⫾ SD.
L.M. Mansky / Virology 307 (2003) 116 –121
119
Fig. 4. Effect of nucleoside reverse transcriptase inhibitors and hydroxyurea on HIV-1 mutant frequencies. Various NRTIs were analyzed along with hydoxyurea for their combined influence on HIV-1 replication in one round of replication. The average mutant frequencies (0.15 mutant/cycle for no drug) were determined from 3 replicate experiments ⫾ SD.
and ddI also led to an increase in the virus mutant frequency (i.e., 5-fold) that best fits an additive model (i.e., 2 ⫹ 2.8 ⫽ 4.8), rather than a multiplicative model (i.e., 2 ⫻ 2.8 ⫽ 5.6), or a synergistic model (significantly higher than 5.6-fold). The results with combined AZT and 3TC dual treatment led to a 6-fold increase in the odds of recovering virus mutants, which best fits an additive model (i.e., 4 ⫹ 2 ⫽ 6). Effect of NRTIs and hydroxyurea on HIV-1 mutant frequencies Hydroxyurea has been previously shown to inhibit HIV-1 replication, alter nucleotide pools, and increase HIV-1 mutant frequencies (Julias and Pathak, 1998; Lori et al., 1997; Mansky et al., 2002; Meyerhans et al., 1994). Studies have indicated that combinations of hydroxyurea and one NRTI can be effective in inhibiting HIV-1 replication (Biron et al., 1996). In particular, ddI and hydroxyurea could be a relatively inexpensive alternative to ART for treatment of HIV-1 infection in underdeveloped countries. The potential interplay between various NRTIs with hydroxyurea on HIV-1 mutant frequencies was analyzed. Based upon the effects of the individual drugs on virus mutant frequencies and infectivities, a hydroxyurea concentration of 0.5 mM was used along with a 0.05 M concentration of either AZT, 3TC, or ddI. For each drug alone, these concentrations had no significant effect on HIV-1 mutant frequencies (Fig. 4). However, when 0.5 mM hydroxyurea was combined with either 0.05 M AZT or 0.05 M 3TC, the resulting virus mutant frequencies were 3.5-
fold higher than the virus mutant frequency observed during virus replication in the absence of drug. Furthermore, the combination of 0.5 mM hydroxyurea with 0.05 M ddI resulted in a 6-fold increase in the virus mutant frequency. Since the drug concentrations used had no effect on the virus mutant frequency alone, the significant changes in the virus mutant frequency from replication in the presence of the different drug combinations was not likely due to additive or multiplicative effects. Therefore, the observed increases in the virus mutant frequency observed with combinations of hydroxyurea and either AZT, 3TC or ddI may be best described with a synergistic model. Further experiments will verify what model is the most appropriate for describing these drug interactions on HIV-1 mutant frequencies.
Discussion This is the first report of antiretroviral drugs acting together to increase HIV-1 mutant frequencies. The mechanisms for how combinations of NRTIs or one NRTI and hydroxyurea increase virus mutant frequencies are currently being investigated. Hypotheses proposed to explain these increased mutant frequencies include: 1) The drugs alter nucleotide pools; 2) The drugs are incorporated into plusstrand DNA and may result in discontinuous DNA synthesis of viral DNAs with proper ends that integrate with subsequent error-prone repair by the host cell; and 3) The drugs
120
L.M. Mansky / Virology 307 (2003) 116 –121
may noncatalytically bind to RT and cause a conformational change that influences enzyme fidelity (Julias et al., 1997). Hydroxyurea is a well-documented drug that alters intracellular dNTP pools. Hydroxyurea alters dNTP pools by inhibiting ribonucleotide reductase and depleting all dNTPs and may also influence DNA repair by increasing the sensitivity of cells to UV irradiation and to other mutagens. The data presented in this report suggest that hydroxyurea can increase the odds of virus mutants during virus replication in the presence of hydroxyurea and an NRTI by dNTP pool depletion. This is presently being further investigated. There is an interest in using combination therapy of hydoxyurea and ddI as an inexpensive treatment for HIV-1 infection that could be more widely used than other more expensive drug combinations in underdeveloped countries. The data presented here suggest that the higher virus mutant frequencies could increase the odds of the selection of drug-resistant viruses and the evolution of drug resistance. Studies to test this hypothesis are currently in progress. The effect of NRTIs on nucleotide pools has not been extensively studied in different cell lines or in primary lymphocytes and macrophages. It is plausible that these drugs, as well as hydroxyurea, could also influence nucleotide pools in particular cell types. Nucleotide pools are believed to be lower in nondividing cells such as macrophages, so the effects described in this report could be greater in macrophages. Depletion of dNTP pools by hydroxyurea could also increase the uptake and phosphorylation of NRTIs by a positive feedback of the nucleoside salvage pathways. The impact of an altered virus mutation rate on HIV-1 diversity is dependent on size of the virus population. Deterministic models have been used for predicting the effects of mutation and selection on HIV-1 populations (Coffin, 1995; Preston, 1997), while stochastic models have been suggested as being more appropriate (Leigh Brown, 1997). For HIV-1 replication in solid lymphoid tissue, a simple metapopulation model for HIV-1 replication shows that the combination of founder effects and subpopulation turnover can result in an effective population size much lower than the actual population size (Frost et al., 2001). This lower population size could contribute to genetic drift being important in HIV-1 evolution despite a large number of infected cells. The mutagenic outcome of anti-HIV chemotherapy would be predicted to be significant in solid tissue.
Materials and methods Retroviral vectors and expression plasmids The expression cassette in the HIV vector used in these studies is shown in Fig. 1A. The vector cassette containing the lacZ gene, an internal ribosomal entry site (IRES) element, and the neomycin phosphotransferase gene (neo) was introduced into pGEM NL4-3 (full length molecular clone of NL4-3) to create pHIVLacZ-IRES-neo. In order to pro-
duce vector virus, the HIV vector was complemented in trans with a HIV-1 gag-pol expression plasmid, an amphotropic murine leukemia virus env expression plasmid, and a Vpr expression plasmid derived from pAS1B (Mansky et al., 2000). Transfections, infections, and cocultivations The COS-1 and HeLa cell lines used were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in Dulbecco’s modified Eagle’s medium containing 10% calf serum or 10% fetal bovine serum, respectively. HIV-1 vector and expression plasmids were transfected into HeLa cells by using Superfect (Qiagen). HeLa cells were infected in the presence of Polybrene. Infection of HeLa target cells was also done by cocultivation of virus-producing cells with target cells. The influence of the antiretroviral drugs on HIV-1 mutant frequencies was determined by postinfection treatment of cells with drug. Postinfection treatment refers to maintaining HeLa target cells in medium supplemented with drug for 2 h before cocultivation and continued until 24 h after cocultivation. Postinfection treatment with drug influences the HIV-1 mutant frequency only during reverse transcription (Mansky and Bernard, 2000). Experimental protocol for generating a single round HIV-1 vector replication The experimental protocol developed to generate a single round of HIV-1 vector replication is shown in Fig. 1B. In this protocol, HeLa cells containing the HIV vector provirus were created by transiently tranfecting the HIV vector and helper plasmids into COS-1 cells, harvesting virus 2 days posttransfection, and infecting HeLa cells. G418-resistant clones were isolated and characterized for the presence of single proviruses. The cells were also stained with X-gal to ensure that no mutations had occurred in the lacZ gene. Selected clones were then used to generate a single round of HIV-1 vector replication. HeLa cell clones with single integrated proviruses were transiently transfected with helper plasmids and were treated with mitomycin C 48 h posttransfection, and then mixed with fresh HeLa cells. G418-resistant cells resulting from virus infection of fresh HeLa cells were selected and cells were then stained with X-gal. The ratio of white plus light-blue stained colonies to total colonies observed provided a forward mutant frequency. Cocultivation of virus-producing cells with permissive target cells was used because it increased the number of infected cells for analysis of mutant frequencies without influencing the rate at which mutations occurred during the one round of replication. In each experiment, similar numbers of colonies were screened for control and experimental samples. Titers for control experiments (wt RT in the absence of drug) were typically 500 –1000 CFU per 5 ⫻ 105 target cells.
L.M. Mansky / Virology 307 (2003) 116 –121
Acknowledgments I thank L. Gajary for technical assistance and D. Pearl for helpful discussions. This research was supported by Public Health Service Grant GM56615.
References Bangsberg, D.R., Hecht, F.M., Charlebois, E.D., Zolopa, A.R., Holodniy, M., Sheiner, L., Bamberger, J.D., Chesney, M.A., Moss, A., 2000. Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population. AIDS 14, 357–366. Biron, F., Lucht, F., Peyramond, D., Fresard, A., Vallet, T., Nugier, F., Grange, J., Malley, S., Hamedi-Sangsari, F., Vila, J., 1996. Pilot clinical trial of the combination of hydroxyurea and didanosine in HIV-1 infected individuals. Antiviral Res. 29, 111–113. Coffin, J., 1995. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267, 483– 489. Frost, S.D.W., Dumaurier, M.-J., Wain-Hobson, S., Leigh Brown, A.J., 2001. Genetic drift and within-host metapopulation dynamics of HIV-1 infection. Proc. Natl. Acad. Sci. USA 98, 6975– 6980. Garcia-Lerma, J.G., Nidtha, S., Blumoff, K., Weinstock, H., Heneine, W., 2001. Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons. Proc. Natl. Acad. Sci. USA 98, 13907–13912. Julias, J.G., Kim, T., Arnold, G., Pathak, V.K., 1997. The antiretrovirus drug 3⬘-azido-3⬘-deoxythymidine increases the retrovirus mutation rate. J. Virol. 71, 4254 – 4263. Julias, J.G., Pathak, V.K., 1998. Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate. J. Virol. 72, 7941–7949. Leigh Brown, A.J., 1997. Analysis of HIV-1 env gene sequences reveals evidence for a low effective number in the viral population. Proc. Natl. Acad. Sci. USA 94, 1862–1865. Lori, F., Malykh, A.G., Foli, A., Maserati, R., De Antoni, A., Minoli, L., Padrini, D., Degli Antoni, A., Barchi, E., Jessen, H., Wainberg, M.A., Gallo, R.C., Lisziewicz, J., 1997. Combination of a drug targeting the
121
cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance. AIDS Res. Hum. Retrovir. 13, 1403–1409. Mansky, L.M., 1996. Forward mutation rate of human immunodeficiency virus type 1 in a T-lymphoid cell line. AIDS Res. Hum. Retroviruses 12, 307–314. Mansky, L.M., Bernard, L.C., 2000. 3⬘-azido-3⬘-deoxythymidine (AZT) and AZT-resistant reverse transcriptase can increase the in vivo mutation rate of human immunodeficiency type 1. J. Virol. 74, 9532–9539. Mansky, L.M., Cunningham, K.S., 2000. Virus mutators and antimutators: roles in evolution, pathogenesis and emergence. Trends in Genetics 16, 512–517. Mansky, L.M., Pearl, D.K., Gajary, L.C., 2002. Combination of drugs and drug-resistant reverse transcriptase results in a multiplicative increase of human immunodeficiency virus type 1 mutant frequencies. J. Virol. 76, 9253–9259. Mansky, L.M., Preveral, S., Selig, L., Benarous, R., Benichou, S., 2000. The interaction of Vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate. J. Virol. 74, 7039 –7047. Mansky, L.M., Temin, H.M., 1995. Lower in vivo mutation rate of human immunodeficiency virus type 1 than predicted from the fidelity of purified reverse transcriptase. J. Virol. 69, 5087–5094. Meyerhans, A., Vartanian, J.-P., Hultgren, C., Plikat, U., Karlsson, A., Wang, L., Eriksson, S., Wain-Hobson, S., 1994. Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools. J. Virol. 68, 535–540. Preston, B.D., 1997. Reverse transcriptase fidelity and HIV-1 variation. Science 275, 228 –231. Richman, D.D., 2001. HIV chemotherapy. Nature 410, 995–1001. Richman, D.D., Havlir, D.V., Corbeil, J., Looney, D., Ignacio, C.C., Spector, S.A., Sullivan, J., Cheeseman, S., Barringer, K., Pauletti, D., 1994. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68, 1660 –1666. Schuurman, R., Nijhuis, M., van Leeuwen, R., Schipper, P., de Jong, D., Collis, P., Danner, S.A., Mulder, J., Loveday, C., Christopherson, C., 1995. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J. Infect Dis. 6 (Suppl 3), 25– 44.
Virology 307 (2003) 116 –121
www.elsevier.com/locate/yviro
Mutagenic outcome of combined antiviral drug treatment during human immunodeficiency virus type 1 replication Louis M. Mansky* Department of Molecular Virology, Immunology, and Medical Genetics, Center for Retrovirus Research, and Comprehensive Cancer Center, Ohio State University Medical Center, Columbus, OH 43210, USA Received 16 August 2002; returned to author for revision 2 October 2002; accepted 14 October 2002
Abstract The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy (ART) is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. To assess the impact of combination therapy on HIV-1 mutagenesis, the mutagenic outcome of combined drug treatment was determined with several different RT drug combinations. Significant increases in HIV-1 mutant frequencies were observed with combinations of nucleoside RT inhibitors as well as in combinations where nucleoside inhibitors were used along with hydroxyurea, a drug known to deplete nucleotide pools in cells. This indicates that combinations of RT drugs can act together to further increase HIV-1 mutant frequencies, which could have important implications for virus population dynamics and could compromise drug therapy regimens. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Nucleoside; Polymerase; Evolution; Drug; Mutagenesis
Introduction The treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals with antiretroviral drugs including reverse transcriptase (RT) and protease inhibitors in a combination therapy (called potent antiretroviral therapy, or ART) has significantly reduced the rate of HIV and AIDS-related morbidity and mortality (Richman, 2001). However, a problem with these therapies is that they can be suboptimal, due in some cases to a lack of patient compliance to drug administration, and allow for the selection of drug-resistant viruses (Bangsberg et al., 2000). Antiviral drugs have been shown to increase virus mutant frequencies of retroviruses and RNA viruses (Mansky and Bernard, 2000; Mansky and Cunningham, 2000). Since the in vivo mutation rate for HIV-1 has been previously determined to be very high (i.e., 4 ⫻ 10⫺5 mutations per target bp per
* Corresponding author. Department of Molecular Virology, Immunology, and Medical Genetics, 2078 Graves Hall, 333 West 10th Ave., Columbus, OH 43210. Fax: ⫹1-614-292-9805. E-mail address: [email protected] (L.M. Mansky).
replication cycle) (Mansky, 1996; Mansky and Temin, 1995), the increased virus mutant frequency in the presence of the drugs could lead to a rapid rate of selection and fixation of mutations that confer drug resistance (Richman et al., 1994; Schuurman et al., 1995). These drug-resistant viruses can readily reside in latently infected cells, which further complicates subsequent drug treatment regimens during the life of the infected individual. When drug-resistance mutations accumulate, drug susceptibility diminishes and reduces the potency of the components of ART. The continued replication in the presence of drug will select for even greater levels of resistance and typically leads to crossresistance to drugs of the same class. Transmission of HIV-1 with reduced susceptibility to antiretroviral drugs may further compromise the efficacy of drug therapy (Garcia-Lerma et al., 2001). In this study, the mutagenic outcome of various drug combinations on HIV-1 replication was analyzed. It was found that virus replication in the presence of nucleoside reverse transcriptase inhibitors (NRTIs) combinations led to an additive increase in virus mutant frequencies. Virus replication in the presence of drug combinations consisting of an NRTI and hydroxyurea,
0042-6822/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0042-6822(02)00069-7
L.M. Mansky / Virology 307 (2003) 116 –121
117
Fig. 1. Assay system used to analyze virus mutant frequencies in vivo during one round of HIV-1 replication using the lacZ gene as a mutation target. A. HIV-1 vector used to measure virus mutant frequencies. The proviral DNA form of the vector is shown. The large black rectangular boxes are the long terminal repeats. The small gray box is the SV40 promoter. The lacZ gene, internal ribosomal entry site (IRES) sequence, and the neo gene are indicated. B. Single-cycle replication assay for mutant frequencies. HeLa cell clones with single integrated vector proviruses were transiently transfected with helper plasmids and the produced virus was used to infect fresh HeLa cells that were treated with drug postinfection (see materials and methods). G418-resistant cells resulting from virus infection of fresh HeLa cells were selected and cells were then stained with X-gal. The ratio of white plus light-blue stained colonies to total colonies was used to determine the forward mutant frequency.
a drug known to deplete dNTP pools in cells, also increased virus mutant frequencies. This indicates that combination drug therapy increases HIV-1 mutant frequencies, which could have important implications for long-term administration of anti-HIV chemotherapy.
Results Dose dependent effect of dd1 on HIV-1 mutant frequencies Various concentrations of ddI, ranging from 0.1 M to 0.8 M, were tested for their effects on virus mutant frequencies using a postinfection treatement strategy (see materials and methods) (Fig. 1). There was a corresponding increase in virus mutant frequency observed (Fig. 2). This indicates that ddI influences the virus mutant frequency in a dose-dependent manner. A dose-dependent relationship between increased drug concentration and increased virus mutant frequency has been previously observed for AZT and 3TC (Mansky and Bernard, 2000), as well as for hydroxyurea (Mansky et al., 2002). The maximum increase in virus mutant frequency in the presence of ddI was 6-fold higher
than the virus mutant frequency during replication in the absence of the drug (P ⬍ 0.0001). Additive effect of NRTI combinations on HIV-1 mutant frequencies Various combinations of NRTIs were tested for their ability to interact together to alter HIV-1 mutant frequencies during virus replication in a manner that was significantly different than observed with individual drugs alone. The drug combinations tested included AZT and 3TC, AZT and ddI, and finally 3TC and ddI. The rationale for testing these combinations was that at least two of these NRTIs are typically used in ART. Control experiments were done with each drug individually to assess the potential interplay between drug combinations in altering virus mutant frequencies. Since each drug individually increased the odds of virus mutants (Fig. 3), it was hypothesized that combined drug treatments would further increase these odds and result in an increase of virus mutant frequencies. Several different outcomes could be envisioned. The drugs could act in an additive manner, a multiplicative manner, or a synergistic manner. In each of the NRTI combinations tested, an additive increase in virus mutant frequencies was observed (Fig.
118
L.M. Mansky / Virology 307 (2003) 116 –121
Fig. 2. Dose-dependent effect of ddI on HIV-1 mutant frequencies. Various concentrations of ddI were analyzed for their influence on HIV-1 replication in one round of replication. Mutant frequencies were determined as described in Fig. 1. The average mutant frequencies (0.15 mutant/cycle at 0 M ddI) were determined from 3 replicate experiments ⫾ SD.
3). In particular, virus replication in the presence of AZT or ddI alone led to 4-fold and 2.8-fold increases in the odds of recovering virus mutants, respectively. However, during HIV-1 replication in the presence of both AZT and ddI, the virus mutant frequency was 6.5-fold higher than observed in
the absence of drug. This 6.5-fold increase in mutant frequency best fits an additive model (i.e., 4 ⫹ 2.8 ⫽ 6.8) versus that of a multiplicative model (i.e., 4 ⫻ 2.8 ⫽ 11.2) or a synergistic model (significantly higher than 11.2-fold). As with the AZT/ddI combination, the combination of 3TC
Fig. 3. Additive effect of nucleoside reverse transcriptase inhibitor (NRTI) combinations on HIV-1 mutant frequencies. Various combinations of NRTIs were analyzed for their influence on HIV-1 replication in one round of replication. The average mutant frequencies (0.14 mutant/cycle for no drug) were determined from 3 replicate experiments ⫾ SD.
L.M. Mansky / Virology 307 (2003) 116 –121
119
Fig. 4. Effect of nucleoside reverse transcriptase inhibitors and hydroxyurea on HIV-1 mutant frequencies. Various NRTIs were analyzed along with hydoxyurea for their combined influence on HIV-1 replication in one round of replication. The average mutant frequencies (0.15 mutant/cycle for no drug) were determined from 3 replicate experiments ⫾ SD.
and ddI also led to an increase in the virus mutant frequency (i.e., 5-fold) that best fits an additive model (i.e., 2 ⫹ 2.8 ⫽ 4.8), rather than a multiplicative model (i.e., 2 ⫻ 2.8 ⫽ 5.6), or a synergistic model (significantly higher than 5.6-fold). The results with combined AZT and 3TC dual treatment led to a 6-fold increase in the odds of recovering virus mutants, which best fits an additive model (i.e., 4 ⫹ 2 ⫽ 6). Effect of NRTIs and hydroxyurea on HIV-1 mutant frequencies Hydroxyurea has been previously shown to inhibit HIV-1 replication, alter nucleotide pools, and increase HIV-1 mutant frequencies (Julias and Pathak, 1998; Lori et al., 1997; Mansky et al., 2002; Meyerhans et al., 1994). Studies have indicated that combinations of hydroxyurea and one NRTI can be effective in inhibiting HIV-1 replication (Biron et al., 1996). In particular, ddI and hydroxyurea could be a relatively inexpensive alternative to ART for treatment of HIV-1 infection in underdeveloped countries. The potential interplay between various NRTIs with hydroxyurea on HIV-1 mutant frequencies was analyzed. Based upon the effects of the individual drugs on virus mutant frequencies and infectivities, a hydroxyurea concentration of 0.5 mM was used along with a 0.05 M concentration of either AZT, 3TC, or ddI. For each drug alone, these concentrations had no significant effect on HIV-1 mutant frequencies (Fig. 4). However, when 0.5 mM hydroxyurea was combined with either 0.05 M AZT or 0.05 M 3TC, the resulting virus mutant frequencies were 3.5-
fold higher than the virus mutant frequency observed during virus replication in the absence of drug. Furthermore, the combination of 0.5 mM hydroxyurea with 0.05 M ddI resulted in a 6-fold increase in the virus mutant frequency. Since the drug concentrations used had no effect on the virus mutant frequency alone, the significant changes in the virus mutant frequency from replication in the presence of the different drug combinations was not likely due to additive or multiplicative effects. Therefore, the observed increases in the virus mutant frequency observed with combinations of hydroxyurea and either AZT, 3TC or ddI may be best described with a synergistic model. Further experiments will verify what model is the most appropriate for describing these drug interactions on HIV-1 mutant frequencies.
Discussion This is the first report of antiretroviral drugs acting together to increase HIV-1 mutant frequencies. The mechanisms for how combinations of NRTIs or one NRTI and hydroxyurea increase virus mutant frequencies are currently being investigated. Hypotheses proposed to explain these increased mutant frequencies include: 1) The drugs alter nucleotide pools; 2) The drugs are incorporated into plusstrand DNA and may result in discontinuous DNA synthesis of viral DNAs with proper ends that integrate with subsequent error-prone repair by the host cell; and 3) The drugs
120
L.M. Mansky / Virology 307 (2003) 116 –121
may noncatalytically bind to RT and cause a conformational change that influences enzyme fidelity (Julias et al., 1997). Hydroxyurea is a well-documented drug that alters intracellular dNTP pools. Hydroxyurea alters dNTP pools by inhibiting ribonucleotide reductase and depleting all dNTPs and may also influence DNA repair by increasing the sensitivity of cells to UV irradiation and to other mutagens. The data presented in this report suggest that hydroxyurea can increase the odds of virus mutants during virus replication in the presence of hydroxyurea and an NRTI by dNTP pool depletion. This is presently being further investigated. There is an interest in using combination therapy of hydoxyurea and ddI as an inexpensive treatment for HIV-1 infection that could be more widely used than other more expensive drug combinations in underdeveloped countries. The data presented here suggest that the higher virus mutant frequencies could increase the odds of the selection of drug-resistant viruses and the evolution of drug resistance. Studies to test this hypothesis are currently in progress. The effect of NRTIs on nucleotide pools has not been extensively studied in different cell lines or in primary lymphocytes and macrophages. It is plausible that these drugs, as well as hydroxyurea, could also influence nucleotide pools in particular cell types. Nucleotide pools are believed to be lower in nondividing cells such as macrophages, so the effects described in this report could be greater in macrophages. Depletion of dNTP pools by hydroxyurea could also increase the uptake and phosphorylation of NRTIs by a positive feedback of the nucleoside salvage pathways. The impact of an altered virus mutation rate on HIV-1 diversity is dependent on size of the virus population. Deterministic models have been used for predicting the effects of mutation and selection on HIV-1 populations (Coffin, 1995; Preston, 1997), while stochastic models have been suggested as being more appropriate (Leigh Brown, 1997). For HIV-1 replication in solid lymphoid tissue, a simple metapopulation model for HIV-1 replication shows that the combination of founder effects and subpopulation turnover can result in an effective population size much lower than the actual population size (Frost et al., 2001). This lower population size could contribute to genetic drift being important in HIV-1 evolution despite a large number of infected cells. The mutagenic outcome of anti-HIV chemotherapy would be predicted to be significant in solid tissue.
Materials and methods Retroviral vectors and expression plasmids The expression cassette in the HIV vector used in these studies is shown in Fig. 1A. The vector cassette containing the lacZ gene, an internal ribosomal entry site (IRES) element, and the neomycin phosphotransferase gene (neo) was introduced into pGEM NL4-3 (full length molecular clone of NL4-3) to create pHIVLacZ-IRES-neo. In order to pro-
duce vector virus, the HIV vector was complemented in trans with a HIV-1 gag-pol expression plasmid, an amphotropic murine leukemia virus env expression plasmid, and a Vpr expression plasmid derived from pAS1B (Mansky et al., 2000). Transfections, infections, and cocultivations The COS-1 and HeLa cell lines used were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in Dulbecco’s modified Eagle’s medium containing 10% calf serum or 10% fetal bovine serum, respectively. HIV-1 vector and expression plasmids were transfected into HeLa cells by using Superfect (Qiagen). HeLa cells were infected in the presence of Polybrene. Infection of HeLa target cells was also done by cocultivation of virus-producing cells with target cells. The influence of the antiretroviral drugs on HIV-1 mutant frequencies was determined by postinfection treatment of cells with drug. Postinfection treatment refers to maintaining HeLa target cells in medium supplemented with drug for 2 h before cocultivation and continued until 24 h after cocultivation. Postinfection treatment with drug influences the HIV-1 mutant frequency only during reverse transcription (Mansky and Bernard, 2000). Experimental protocol for generating a single round HIV-1 vector replication The experimental protocol developed to generate a single round of HIV-1 vector replication is shown in Fig. 1B. In this protocol, HeLa cells containing the HIV vector provirus were created by transiently tranfecting the HIV vector and helper plasmids into COS-1 cells, harvesting virus 2 days posttransfection, and infecting HeLa cells. G418-resistant clones were isolated and characterized for the presence of single proviruses. The cells were also stained with X-gal to ensure that no mutations had occurred in the lacZ gene. Selected clones were then used to generate a single round of HIV-1 vector replication. HeLa cell clones with single integrated proviruses were transiently transfected with helper plasmids and were treated with mitomycin C 48 h posttransfection, and then mixed with fresh HeLa cells. G418-resistant cells resulting from virus infection of fresh HeLa cells were selected and cells were then stained with X-gal. The ratio of white plus light-blue stained colonies to total colonies observed provided a forward mutant frequency. Cocultivation of virus-producing cells with permissive target cells was used because it increased the number of infected cells for analysis of mutant frequencies without influencing the rate at which mutations occurred during the one round of replication. In each experiment, similar numbers of colonies were screened for control and experimental samples. Titers for control experiments (wt RT in the absence of drug) were typically 500 –1000 CFU per 5 ⫻ 105 target cells.
L.M. Mansky / Virology 307 (2003) 116 –121
Acknowledgments I thank L. Gajary for technical assistance and D. Pearl for helpful discussions. This research was supported by Public Health Service Grant GM56615.
References Bangsberg, D.R., Hecht, F.M., Charlebois, E.D., Zolopa, A.R., Holodniy, M., Sheiner, L., Bamberger, J.D., Chesney, M.A., Moss, A., 2000. Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population. AIDS 14, 357–366. Biron, F., Lucht, F., Peyramond, D., Fresard, A., Vallet, T., Nugier, F., Grange, J., Malley, S., Hamedi-Sangsari, F., Vila, J., 1996. Pilot clinical trial of the combination of hydroxyurea and didanosine in HIV-1 infected individuals. Antiviral Res. 29, 111–113. Coffin, J., 1995. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267, 483– 489. Frost, S.D.W., Dumaurier, M.-J., Wain-Hobson, S., Leigh Brown, A.J., 2001. Genetic drift and within-host metapopulation dynamics of HIV-1 infection. Proc. Natl. Acad. Sci. USA 98, 6975– 6980. Garcia-Lerma, J.G., Nidtha, S., Blumoff, K., Weinstock, H., Heneine, W., 2001. Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons. Proc. Natl. Acad. Sci. USA 98, 13907–13912. Julias, J.G., Kim, T., Arnold, G., Pathak, V.K., 1997. The antiretrovirus drug 3⬘-azido-3⬘-deoxythymidine increases the retrovirus mutation rate. J. Virol. 71, 4254 – 4263. Julias, J.G., Pathak, V.K., 1998. Deoxyribonucleoside triphosphate pool imbalances in vivo are associated with an increased retroviral mutation rate. J. Virol. 72, 7941–7949. Leigh Brown, A.J., 1997. Analysis of HIV-1 env gene sequences reveals evidence for a low effective number in the viral population. Proc. Natl. Acad. Sci. USA 94, 1862–1865. Lori, F., Malykh, A.G., Foli, A., Maserati, R., De Antoni, A., Minoli, L., Padrini, D., Degli Antoni, A., Barchi, E., Jessen, H., Wainberg, M.A., Gallo, R.C., Lisziewicz, J., 1997. Combination of a drug targeting the
121
cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance. AIDS Res. Hum. Retrovir. 13, 1403–1409. Mansky, L.M., 1996. Forward mutation rate of human immunodeficiency virus type 1 in a T-lymphoid cell line. AIDS Res. Hum. Retroviruses 12, 307–314. Mansky, L.M., Bernard, L.C., 2000. 3⬘-azido-3⬘-deoxythymidine (AZT) and AZT-resistant reverse transcriptase can increase the in vivo mutation rate of human immunodeficiency type 1. J. Virol. 74, 9532–9539. Mansky, L.M., Cunningham, K.S., 2000. Virus mutators and antimutators: roles in evolution, pathogenesis and emergence. Trends in Genetics 16, 512–517. Mansky, L.M., Pearl, D.K., Gajary, L.C., 2002. Combination of drugs and drug-resistant reverse transcriptase results in a multiplicative increase of human immunodeficiency virus type 1 mutant frequencies. J. Virol. 76, 9253–9259. Mansky, L.M., Preveral, S., Selig, L., Benarous, R., Benichou, S., 2000. The interaction of Vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 in vivo mutation rate. J. Virol. 74, 7039 –7047. Mansky, L.M., Temin, H.M., 1995. Lower in vivo mutation rate of human immunodeficiency virus type 1 than predicted from the fidelity of purified reverse transcriptase. J. Virol. 69, 5087–5094. Meyerhans, A., Vartanian, J.-P., Hultgren, C., Plikat, U., Karlsson, A., Wang, L., Eriksson, S., Wain-Hobson, S., 1994. Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools. J. Virol. 68, 535–540. Preston, B.D., 1997. Reverse transcriptase fidelity and HIV-1 variation. Science 275, 228 –231. Richman, D.D., 2001. HIV chemotherapy. Nature 410, 995–1001. Richman, D.D., Havlir, D.V., Corbeil, J., Looney, D., Ignacio, C.C., Spector, S.A., Sullivan, J., Cheeseman, S., Barringer, K., Pauletti, D., 1994. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68, 1660 –1666. Schuurman, R., Nijhuis, M., van Leeuwen, R., Schipper, P., de Jong, D., Collis, P., Danner, S.A., Mulder, J., Loveday, C., Christopherson, C., 1995. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J. Infect Dis. 6 (Suppl 3), 25– 44.