5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine

• Type of Material: Article
• Authors: Jonathan M.O. Rawson, University of Minnesota; Michele B. Daly, Emory University; Jiashu Xie, University of Minnesota; Christine L. Clouser, University of Minnesota; Sean R. Landman, University of Minnesota; Cavan S. Reilly, University of Minnesota; Laurent Bonnac, University of Minnesota; Baek Kim, Emory University; Steven E. Patterson, University of Minnesota; Louis M. Mansky, University of Minnesota
• Language: English
• Date: 2016-04-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2016, American Society for Microbiology. All Rights Reserved.
• Final Published Version (URL): http://dx.doi.org/10.1128/AAC.03084-15
• Title of Journal or Parent Work: Antimicrobial Agents and Chemotherapy
• ISSN: 0066-4804
• Volume: 60
• Issue: 4
• Start Page: 2318
• End Page: 2325
• Grant/Funding Information: This research was funded by fellowships (to J.M.O.R. and S.R.L.) from the University of Minnesota Graduate School.; National Institutes of Health (NIH) provided funding to Jonathan Rawson under grant number F31 DA035720.; National Institutes of Health (NIH) provided funding to Jonathan M. O. Rawson under grant number T32 AI83196.; National Institutes of Health (NIH) provided funding to Louis M. Mansky under grant number R01 GM105876.; HHS
• Keywords: ANTI-HIV-1 ACTIVITY; DNA-SYNTHESIS; Science & Technology; SMALL-MOLECULE INHIBITION; Pharmacology & Pharmacy; MACROPHAGES; TEMPLATE; RIBONUCLEOTIDE INCORPORATION; DNTP BINDING; LETHAL MUTAGENESIS; TYPE-1 REVERSE-TRANSCRIPTASE; VIF; Microbiology; Life Sciences & Biomedicine
• Research Categories: Biology, Virology; Biology, Microbiology; Health Sciences, Pharmacology

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-CTP in vitro at 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.