5-Azacytidine enhances the mutagenesis of HIV-1 by reduction to 5-aza-2'-deoxycytidine

Jonathan M.O. Rawson, Michele B. Daly, Jiashu Xie, Christine L Clouser, Sean R. Landman, Cavan S Reilly, Laurent Bonnac, Baek Kim, Steven Patterson, Louis M Mansky

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2318-2325
Number of pages8
JournalAntimicrobial Agents and Chemotherapy
Volume60
Issue number4
DOIs
StatePublished - Apr 1 2016

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decitabine
Azacitidine
Mutagenesis
HIV-1
Reverse Transcription
Ribonucleosides
Cytidine Triphosphate
Mutation
Deoxyribonucleotides
Ribonucleotides
Ribonucleotide Reductases
Retroviridae
Tandem Mass Spectrometry
Liquid Chromatography
2'-deoxycytidine 5'-triphosphate
DNA

Cite this

5-Azacytidine enhances the mutagenesis of HIV-1 by reduction to 5-aza-2'-deoxycytidine. / Rawson, Jonathan M.O.; Daly, Michele B.; Xie, Jiashu; Clouser, Christine L; Landman, Sean R.; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven; Mansky, Louis M.

In: Antimicrobial Agents and Chemotherapy, Vol. 60, No. 4, 01.04.2016, p. 2318-2325.

Research output: Contribution to journalArticle

Rawson, Jonathan M.O. ; Daly, Michele B. ; Xie, Jiashu ; Clouser, Christine L ; Landman, Sean R. ; Reilly, Cavan S ; Bonnac, Laurent ; Kim, Baek ; Patterson, Steven ; Mansky, Louis M. / 5-Azacytidine enhances the mutagenesis of HIV-1 by reduction to 5-aza-2'-deoxycytidine. In: Antimicrobial Agents and Chemotherapy. 2016 ; Vol. 60, No. 4. pp. 2318-2325.
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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.",
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AU - Clouser, Christine L

AU - Landman, Sean R.

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AU - Patterson, Steven

AU - Mansky, Louis M

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