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. 2009 Nov;83(22):11950-8.
doi: 10.1128/JVI.01406-09. Epub 2009 Sep 2.

5-Azacytidine can induce lethal mutagenesis in human immunodeficiency virus type 1

Michael J Dapp  1 , Christine L Clouser, Steven Patterson, Louis M Mansky
Affiliations

5-Azacytidine can induce lethal mutagenesis in human immunodeficiency virus type 1

Michael J Dapp et al. J Virol. 2009 Nov.

Abstract

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2'-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription.

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Figures

FIG. 1.
FIG. 1.
HIV-1 vector for monitoring viral infectivity and mutant frequency in the presence of antiretroviral drugs. An envelope- and Nef-deficient HIV-1 vector was constructed with a gene cassette containing the mouse HSA, an IRES element, and the GFP gene. This vector was cotransfected into 293T cells, along with a HIV envelope expression plasmid for producing vector virus. Forty-eight hours posttransfection, the cell culture supernatants were collected, filtered, and used to infect permissive U373-MAGICXCR4 target cells at an MOI of 0.3. Forty-eight hours postinfection, cells were harvested, stained with a PE-conjugated HSA antibody, and analyzed by flow cytometry. Virus-producing cells or permissive target cells were pretreated with drug or DMSO for 12 h after either transfection or 2 h prior to infection, respectively, and treatment continued 24 h postinfection. FACS, fluorescence-activated cell sorting.
FIG. 2.
FIG. 2.
Concentration-dependent effects of 5-AZC on HIV-1 infectivity and mutant frequency. 293T or U373-MAGICXCR4 cells were treated with the indicated concentrations of compound prior to transfection (late-phase replication) (A and B) or infection (early-phase replication) (C and D). Infected cells (MOI, 0.3) were analyzed by flow cytometry. Only the ribonucleoside 5-AZC had a concentration-dependent effect on viral infectivity in treatment during late-phase replication (A), and this coincided with an increase in the viral mutant frequency (B). (C) A more potent antiviral effect was observed with 5-AZC treatment during early-phase replication. (D) This antiviral activity coincided with a much more dramatic increase in the viral mutant frequency. The data shown are means ± standard deviations of five independent experiments.
FIG. 3.
FIG. 3.
Cytotoxicity of 5-AZC. 293T (A) or U373-MAGICXCR4 (B) cells were treated at the indicated concentrations of 5-AZC and analyzed for cell viability by measuring cellular ATP levels. The data represent the means ± standard deviations of four independent experiments.
FIG. 4.
FIG. 4.
Mutation spectra of GFP gene sequences from vector proviral DNAs. The HSA+/GFP− cell population from infected U373-MAGI cells was sorted from cells treated with 5-AZC (Fig. 1). The sequence results from at least 100 mutants are shown for no drug (A), 5-AZC treatment of permissive target cells (early phase) (B), and 5-AZC treatment of virus-producing cells (late phase) (C). Each GFP gene sequence analyzed is represented by a thin gray horizontal line. The location of a mutation in the GFP gene sequence is indicated by a colored box perpendicular to the line relative to the 5′ end of the 720-bp open reading frame. Transition mutations are represented by either black (purine) or yellow (pyrimidine) rectangular boxes. G-to-C and C-to-G transversion mutations are indicated by red and green rectangular boxes, respectively. Transversion mutations (other than G-to-C and C-to-G) are indicated by blue rectangular boxes. (D) Mutational loads in GFP genes from proviral DNAs. A summary of the mutational loads (all mutation types) in the GFP gene from either no drug, 5-AZC early phase, or 5-AZC late phase is shown, with each diamond representing a proviral GFP sequence. The calculation of the average number of mutations per GFP target gene sequence is shown.
FIG. 5.
FIG. 5.
5-AZC inhibits replication-competent HIV-1. The HIV-1 NL4-3 molecular clone was transfected into 293T cells to produce an infectious virus stock that was used to infect CEM-GFP cells. The cells were treated with 5-AZC at the indicated concentrations. The cells were split every 2 days, and fresh medium and 5-AZC were added. Flow cytometry was used to determine the percentage of infected cells every 2 days. The data represent the means ± standard errors of parallel experiments done in triplicate from 8 days posttreatment and are representative of three independent experiments.
FIG. 6.
FIG. 6.
Model of 5-AZC mutagenesis during minus-strand DNA synthesis in HIV-1 reverse transcription. Ribonucleotide reductase converts 5-AZCDP to 5-aza-dCDP. After the incorporation of 5-aza-dC (dZ) triphosphate into minus-strand viral DNA, a spontaneous cytosine ring opening to a Dimroth intermediate occurs, which allows the base to pair with dC. During integration, 5-aza-dC is excised by DNA repair machinery and replaced with a guanosine, since it base pairs with the cytosine present opposite the abasic site in the plus-strand DNA. When transcribed, the guanosine in the minus strand codes for a cytosine, thereby leading to an overall G-to-C mutation.
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