Authors: Jonathan M.O. Rawson, Michele B. Daly, Jiashu Xie, Christine L. Clouser, Sean R. Landman, Cavan S. Reilly, Laurent Bonnac, Baek Kim, Steven E. Patterson and Louis M. Mansky
Summary:
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-azacytidine-triphosphate, or 5-aza-CTP) or as a deoxyribonucleotide (5-aza-2’-deoxycytidine-triphosphate, or 5-aza-dCTP) during reverse transcription. In order to determine the primary form of 5-aza-C 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, both 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 antiviral effect 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.
Source:
Antimicrobial Agents & Chemotherapy; 02/01/16