The emergence and transmission of drug resistance, as well as the halting of the Merck vaccine trial, emphasize the continual need for the development of novel HIV-1 drugs, as well as a return to fundamental aspects of vaccine development (20
). As an antiviral strategy, lethal mutagenesis offers a novel drug target (i.e., the viral mutation rate) and is likely to have a high barrier to drug resistance. However, little progress has been made in identifying compounds with enough therapeutic potential to be used clinically to promote HIV-1 lethal mutagenesis. The ribonucleoside analog ribavirin is the only clinically approved ribonucleoside identified so far that may act as a lethal mutagen to inhibit viral replication, specifically, that of hepatitis C virus (10
). While ribavirin is not effective against HIV-1, other ribonucleoside analogs, such as 5-AZC, have been shown to have anti-HIV activity in cell culture (3
). However, little is known about its mechanism of action.
In this study, we examined the antiviral activity, mechanism of action, and toxicity of 5-AZC. Since 5-AZC is a ribonucleoside analog, it was hypothesized that its antiviral activity would primarily be attributed to its incorporation into viral RNA and the subsequent increase in the HIV-1 mutation frequency. In support of this, several previous studies have shown that 5-AZC can be incorporated into RNA (6
). One study demonstrated that 5-AZC was a weak competitive inhibitor with a 20-fold-lower affinity than CTP for RNA polymerase II (35
). However, our results show that the most potent antiviral activity of 5-AZC can actually be attributed to its effect on the early phase of HIV-1 replication, which includes reverse transcription. In fact, while 5-AZC increased the HIV-1 mutation frequency in both the late and early phases of HIV-1 replication, it had a greater effect on the early phase of replication. These data suggest that 5-AZC exerts its antiviral activity at both phases of replication through an increase in the mutation frequency. Although 5-AZC led to a modest increase in the mutant frequency, similar increases in mutation rates have been shown to be sufficient to lethally mutagenize other RNA viruses (24
). In fact, the theory of lethal mutagenesis suggests that small increases in viral mutation rates should lead to a disproportionately larger decrease in viral infectivity (13
Both phosphorylation and 2′-OH reduction are prerequisites for 5-AZC's clinical use as a DNA hypomethylating agent (reviewed in references 29
). Similarly, our results suggest that 5-AZC is likely to be phosphorylated and reduced prior to its incorporation into viral DNA by RT. Phosphorylation of 5-AZC is likely performed by uridine-cytidine kinase, as this enzyme is responsible for the phosphorylation of CMP, UMP, and dCMP, as well as many pyrimidine analogs used for cancer and antiviral therapy (28
). Additionally, 5-AZC was shown to be a suitable substrate for uridine-cytidine kinase (37
After phosphorylation, it is inferred that 5-aza-CDP is reduced by cellular ribonucleotide reductase before its incorporation into viral DNA. It has been shown that 10 to 20% of 5-AZC is incorporated into cellular DNA, suggesting a metabolic pathway through ribonucleotide reductase (36
). To further support a role for ribonucleotide reductase in 5-AZC metabolism, the ribonucleotide reductase inhibitor hydroxyurea was found to block the epigenetic hypomethylation activity of 5-AZC in vivo (12
). Furthermore, our mutational data support the conversion of 5-AZC and its incorporation into DNA. Specifically, our data show a significant increase in G-to-C transversion mutations in proviral DNA after target cells were treated with 5-AZC (Fig. and Table ), and a similar increase in G-to-C mutations was reported for cellular DNA exposed to 5-aza-dC (30
). To our knowledge, there have been no biochemical studies that have looked at the interaction between 5-AZCTP (or 5-aza-dCTP) and purified RT; however, it is well established that the DNA polymerase activity of RT is specific for deoxynucleoside triphosphates (dNTPs) by preferentially excluding ribonucleoside triphosphates (rNTPs) from entering the polymerase active site (18
). Moreover, previous reports have shown that DNA polymerase α has similar affinities and rates of incorporation for 5-aza-dCTP and dCTP (2
Based on the data presented here, we propose a model that accounts for 5-AZC's antiviral effect on the early phase of the HIV-1 replication cycle (Fig. ). In this model, 5-AZC is first reduced to 5-aza-dCDP by ribonucleotide reductase. Next, 5-aza-dCTP is incorporated into viral DNA during reverse transcription. Once incorporated into DNA, the 5-aza-cytosine triazine ring (i.e., the base) can undergo a ring-opening step that would enable it to base pair with cytosine (30
). Thus, as shown in Fig. , cytosine would be incorporated into the plus-strand DNA opposite 5-aza-dC. Finally, our model shows that integration is a key step in repairing the 5-AZC-induced mutations. The model proposes that 5-aza-dC is excised by host DNA repair machinery during integration. 5-Aza-dC would be replaced with guanosine, since it can base pair with the cytosine located in the plus-strand DNA opposite the abasic site. When transcribed, the minus-strand DNA then results in viral progeny carrying G-to-C mutations. In contrast, 5-aza-dC could be incorporated into the positive-strand viral DNA across from guanosines present in the minus strand. However, during integration, the DNA repair machinery would likely excise the 5-aza-dC and replace it with a cytosine, which would not lead to a mutation.
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 (more ...)
The use of ribonucleoside analogs as lethal mutagens offers the benefit of being able to predict the type of mutations that give rise to virus lethality (39
). The increase in G-to-C mutations caused by 5-AZC could be predicted based on the base-pairing properties of 5-AZC and 5-aza-dC. Thus, it is possible that ribonucleoside analogs could be designed to specifically target certain nucleotides for mutation. Ribonucleoside analogs may be superior to current HIV-1 drugs in their ability to delay the emergence of drug resistance. For high-level resistance to emerge against ribonucleoside analogs that function like 5-AZC, it is likely that mutations would have to be acquired in both RT and RNA polymerase II. Although RT is likely to accumulate mutations, there is little pressure on RT to select for mutations that would exclude mutagenic nucleoside analogs, since these drugs do not appear to prevent RT-mediated polymerization. This is in contrast to nucleoside analogs, such as azidothymidine, which prevent replication by chain termination and therefore efficiently select for any mutations in RT that restore viral-DNA synthesis.
A limitation to the development of ribonucleosides as potential antiretroviral agents is the relatively high concentrations needed to observe an antiviral effect (Fig. ). However, the high concentrations of 5-AZC shown here may be attributed in part to the cell lines used in this study. A previous meta-analysis study documented that cell lines have up to fivefold-higher concentrations of rNTPs than primary cells and that cellular rNTPs are 10- to 100-fold more abundant that dNTPs (51
). This suggests that during late-phase replication, 5-AZCTP must compete with an intracellular concentration of CTP of 109 to 455 μM. Similarly, dividing cells were found to have intracellular dCTP concentrations ranging from 27 to 50 μM (51
). Because the current study investigated treatment with the ribonucleoside 5-AZC, which has the potential to participate in a number of nucleos(t)ide metabolic pathways, it is difficult to predict the intracellular concentrations of the rNTP and dNTP forms of 5-AZC. Nonetheless, these particular cell lines were chosen because of technical reasons during the virological assays, as well as the sheer number of cells needed for the sequencing experiments.
Differences in dNTP pools in the cell types used here may also account for the discrepancy in IC50
values when the single-cycle assay (Fig. ) is compared to the multiple-round assay (Fig. ), although differences in transport pathways and metabolisms could also play a role. Future studies will be needed to precisely measure intracellular 5-AZCTP and 5-aza-dCTP levels when cells are exposed to ribonucleosides. It may also be of interest to measure endogenous dNTP pool levels when treating cells with nucleoside analogs, because this has been shown to cause alterations in natural dNTP pool levels (52
) and thus may contribute to an increase in the retroviral mutation rate (4
). However, the presence of the specific G-to-C transversion in the presence of 5-AZC seems to argue against this notion (Fig. and Table ).
Since our data support a model in which 5-AZC is converted to the corresponding deoxyribonucleoside triphosphate, it is possible that it could be incorporated into the host genome. This raises concerns about the possible genotoxicity of potential mutagenic ribonucleosides. However, toxicity was not significant at the concentrations required to inhibit viral replication. Additionally, a nucleoside analog currently in development, KP1212/KP1461, induces mutations in viral DNA but does not appear to do so in the host cell genome (24
). It is likely that the host DNA repair machinery is sufficiently effective to eliminate any of these analogs that are incorporated into genomic DNA. However, the novelty of these drugs warrants further investigation into their potential long-term effects.
Previous studies have demonstrated the anti-HIV activity of 5-AZC (3
). Based on the structure of 5-AZC, it was speculated that its antiviral activity was due to its ability to be incorporated into viral RNA during transcription. Here, our data show that while 5-AZC does demonstrate antiviral activity by this mechanism, its more potent anti-HIV activity can be attributed to its reduction to 5-aza-dCTP followed by incorporation into viral DNA during reverse transcription. Thus, 5-AZC inhibits HIV-1 infectivity through its incorporation into both viral RNA and DNA. This incorporation significantly increases the HIV-1 mutation frequency to a point consistent with lethal mutagenesis. Compounds with a similar mechanism of action could represent an important new class of anti-HIV compounds to explore for clinical viability.