5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1

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.

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

“…After 16 passages in drug, we observed an increase in two mutation types for each drug that are consistent with their chemical structures and previous work in other viral systems (15,23,25,28). In contrast, only one of the two expected mutation types was observed in the mutation frequency assay after a single passage in ribavirin or 5-azacytidine.…”

“…In our study of lethal mutagenesis, we used ribavirin, 5-azacytidine, and 5-fluorouracil, each of which is a known mutagen of other RNA viruses (15,23,25,55). These three drugs were selected, in part, due to their activity against a range of RNA viruses as well as differences in the types of mutations that they are known to induce.…”

“…While ribavirin is used clinically for hepatitis C virus and respiratory syncytial virus, its mode of action against these viruses in vivo is less clear (20)(21)(22). Lethal mutagenesis with 5-azacytidine has been demonstrated in HIV-1 and foot-and-mouth disease virus (FMDV) in vitro (23,24). The base analog 5-fluorouracil is processed intracellularly into a nucleoside analog and has demonstrated activity as a lethal mutagen against LCMV, exonucleasedeficient coronaviruses, and FMDV in vitro (24)(25)(26).…”

“…Ribavirin, a guanosine analog, causes an increase in both C-to-U and G-to-A transitions (15), and 5-fluorouracil, which mimics uridine, leads to the accumulation of A-to-G and U-to-C transitions (25). Interestingly, 5-azacytidine, a cytidine analog, is able to induce both C-to-G and G-to-C transversions by virtue of a pyrimidine ring-opening mechanism that allows it to base pair with cytosine (23,28). Ribavirin also has additional mechanisms that may play a role in its antiviral activity.…”

Exploiting Drug Repositioning for Discovery of a Novel HIV Combination Therapy

“…Although there is no evidence that decitabine is mutagenic in humans, there is some evidence that decitabine could be mutagenic in some systems, such a transgenic mouse model where an increase in C:G3G:C transversions in the lacZ transgene was reported (23). Similarly, exposure of HIV type 1 with 5-azacytidine, the ribonucleoside analog of decitabine, increased C:G3G:C mutations (9). The model of the mutagenic lesions identified suggests that decitabine can un- FIG.…”