title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.
abstract: 5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.
authors.affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jonathan M O
authors.initials: JM
authors.lastname: Rawson
authors.affiliation: Center for Drug Discovery, Department of Pediatrics, Emory Center for AIDS Research, Emory University, Atlanta, Georgia, USA.
authors.forename: Michele B
authors.initials: MB
authors.lastname: Daly
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jiashu
authors.initials: J
authors.lastname: Xie
authors.affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Clouser
authors.affiliation: Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Landman
authors.affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Cavan S
authors.initials: CS
authors.lastname: Reilly
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Laurent
authors.initials: L
authors.lastname: Bonnac
authors.affiliation: Center for Drug Discovery, Department of Pediatrics, Emory Center for AIDS Research, Emory University, Atlanta, Georgia, USA.
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Patterson
authors.affiliation: Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, Minnesota, USA Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, USA Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, Minneapolis, Minnesota, USA Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota, USA mansky@umn.edu.
authors.forename: Louis M
authors.initials: LM
authors.lastname: Mansky
mesh_terms: D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D002853:Chromatography, Liquid; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D000077209:Decitabine; D057809:HEK293 Cells; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis / Q000187:drug effects*; D010084:Oxidation-Reduction; D011533:Proviruses / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D048348:Reverse Transcription / Q000187:drug effects; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D003570:Cytidine Triphosphate; C038673:5-aza-2'-deoxycytidine-5'-triphosphate; D000077209:Decitabine; D012264:Ribonucleotide Reductases; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D001374:Azacitidine
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