Journal: Antimicrobial Agents and Chemotherapy

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

doi: 10.1128/AAC.03084-15

Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant (P > 0.05).

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Infection, Purification, Sequencing, Plasmid Preparation, Amplification

Journal: Antimicrobial Agents and Chemotherapy

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

doi: 10.1128/AAC.03084-15

Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar patterns of mutation during HIV-1 replication. Using the Illumina sequencing data, G-to-C and C-to-G transversion frequencies were determined at every individual guanine (124 in total) or cytosine (116 in total) position within the sequences of the five amplicons. Mutation frequencies for each amplicon were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases) and are represented as mutations per base pair (m/bp). 5-Aza-C- and 5-aza-dC-induced mutation frequencies were then plotted against each other for each sequence position, and the resulting data were subjected to linear regression and correlation analyses. Data represent averages for three independent biological replicates. R2 denotes the extent to which the best-fit regression line explains the observed variability in the data; P indicates the significance of the correlation; and m indicates the slope of the best-fit regression line.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Sequencing, Amplification

Journal: Antimicrobial Agents and Chemotherapy

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

doi: 10.1128/AAC.03084-15

Figure Lengend Snippet: 5-Aza-dCTP levels are comparable in cells treated with 5-aza-C or 5-aza-dC. In order to determine the extent to which 5-aza-C is reduced to 5-aza-dC intracellularly, U373-MAGI cells were incubated with varying concentrations (EC25, EC50, or EC75) of 5-aza-C or 5-aza-dC. Cells were collected for analysis 4 h after drug addition, a time corresponding to the expected time of early reverse transcription. LC–MS-MS was then used to determine the relative levels of 5-aza-dCTP and 2′-deoxyriboguanylurea 5′-triphosphate (dRGU-TP). dRGU-TP is the final hydrolysis product of 5-aza-dCTP and is potentially relevant to antiviral activity. Data represent means ± standard deviations for three independent experiments, normalized to the EC75 of 5-aza-dC. N.S., not significant; *, P < 0.05.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

doi: 10.1128/AAC.03084-15

Figure Lengend Snippet: HIV-1 RT incorporates 5-aza-CTP much less efficiently than 5-aza-dCTP in vitro. The relative abilities of HIV-1 RT to incorporate 5-aza-CTP, 5-aza-dCTP, CTP, and dCTP were determined using an in vitro single nucleotide extension assay. The HIV-1 RT was incubated with a radiolabeled primer (18 nt) annealed to a DNA template (19 nt) in the presence of each compound at 5 nM to 500 μM (a 10-fold series, from left to right). Additional control reactions were performed by incubating with all four standard dNTPs (50 μM) (+) or by omitting RT (−). All of the reactions were analyzed on the same gel.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: In Vitro, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

doi: 10.1128/AAC.03084-15

Figure Lengend Snippet: Model of 5-azacytidine- and 5-aza-2′-deoxycytidine-mediated HIV-1 mutagenesis. 5-Aza-C and 5-aza-dC are first transported into the cell by facilitated diffusion through human equilibrative nucleoside transporter 1 (hENT1). 5-Aza-C and 5-aza-dC are then phosphorylated by uridine-cytidine kinase (UCK) and deoxycytidine kinase (dCK), respectively, to the monophosphate forms. Nucleoside monophosphate and diphosphate kinases (NMPK and NDPK) phosphorylate the monophosphate and diphosphate forms, respectively, resulting in the formation of 5-aza-CTP and 5-aza-dCTP. 5-Aza-CTP can be incorporated during the transcription of viral genomic RNA, resulting in C-to-G transversions. 5-Aza-CDP is also reduced to 5-aza-dCDP, ultimately forming 5-aza-dCTP. 5-Aza-dCTP is incorporated during reverse transcription, resulting primarily in G-to-C transversions (reflecting minus-strand incorporation) but also in low levels of C-to-G transversions. The mechanism by which C-to-G transversions are formed is still unclear, but they may be due to plus-strand incorporation of 5-aza-dCTP or minus-strand incorporation of 5-aza-dCTP hydrolysis products.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Diffusion-based Assay