Recent setbacks in the development of an AIDS vaccine, as well as the prevalence of drug-resistant HIV, emphasize the need for new approaches to anti-HIV therapies. To decrease the cost of and time required for the development of novel drugs to treat HIV infection, a number of clinically approved drugs were screened for the ability to inhibit HIV infectivity, including nucleoside analogs and antimetabolites. Since HIV reverse transcriptase is error prone, the virus is highly susceptible to mutations caused by alterations in dNTP pools or by the incorporation of nucleoside analogs that form noncanonical base pairs (4
). The infectivity of HIV and other RNA viruses is significantly reduced with even a modest increase in the mutation rate (19
). This finding has led to the design of novel nucleoside analogs, including 5-hydroxy-2′-deoxycytidine and 5-aza-5,6-dihydro-2′-deoxycytidine (KP-1212), that are incorporated into viral DNA during replication (16
). Although these compounds effectively increase the viral mutation rate, none have been shown to be potent or safe enough to be used clinically.
In this study, we identify a novel strategy for HIV infection treatment that takes advantage of a drug target that has yet to be exploited clinically—the HIV mutation rate. Specifically, we show that HIV infectivity can be synergistically decreased by combining two classes of compounds, (i) a nucleoside analog that forms noncanonical base pairs and (ii) a ribonucleotide reductase inhibitor. Another significant finding was that the antiviral activity of the drug combination can be attributed to an increase in the HIV mutation rate. The most potent drug combination characterized consists of decitabine and gemcitabine, two clinically approved drugs used in cancer treatment. The data shown here demonstrate that when used in combination, decitabine and gemcitabine appear to synergistically decrease HIV infectivity at concentrations well below those used in cancer therapy (14
). Furthermore, a combination of decitabine and gemcitabine increased the HIV mutation frequency by 3.4-fold (Fig. ) compared to that in untreated cells and this increase in the mutation frequency correlated with a 73% reduction in infectivity (Fig. ). Although the increase in the mutation frequency was modest, similar increases have been found to be sufficient to eliminate the infectivity or viability of other viruses, such as FMDV, poliovirus, and HIV (19
). The hypothesis that combination drug therapy induces viral mutations was further supported by sequencing data, which showed a striking difference between the mutation spectra of viruses exposed to combination drug therapy and unexposed viruses. A model is proposed in Fig. to account for the observed changes in the mutation spectrum when decitabine is incorporated into viral DNA. These findings suggest that combination drug therapy decreases infectivity by increasing the mutation rate. This is further supported by data showing that similar antiviral effects were observed when decitabine was combined with a different ribonucleotide reductase inhibitor, hydroxyurea. Hydroxyurea has previously been shown to have anti-HIV activity and acts synergistically with chain terminators such as dideoxyinosine (11
). Although hydroxyurea has been used clinically to treat HIV infection, its use is not favored because of its adverse effects. While it was expected that other ribonucleotide reductase inhibitors would have the same antiviral effects as that seen with hydroxyurea and gemcitabine, this was not supported by our data. This may be related to the specific dNTP pools that are altered by each of the different compounds or cellular processes that compensate for alterations in dNTP pools.
FIG. 7. Model of how decitabine acts as a viral mutagen to induce G-to-C mutations during minus-strand viral DNA synthesis. Positive-stranded viral RNA is reverse transcribed into double-stranded viral DNA. Decitabine (D*) is incorporated in place of (more ...)
As a clinical therapy, lethal mutagenesis has been met with hesitation, primarily because of concerns related to the cytotoxicity of the compounds, the high concentrations of mutagen required, and the potential side effects associated with the long-term use of these compounds (25
). However, the concentrations of decitabine and gemcitabine used here are well below the concentrations used in cancer therapy (31
), suggesting that any adverse effects of these drugs should also be minimized. Although our data indicate that decitabine can act as a mutagenic nucleoside to inhibit HIV replication, clinically, decitabine is not known to be mutagenic. Instead, decitabine is thought to work by covalently binding to DNA methyltransferase I, irreversibly inhibiting the enzyme and decreasing DNA methylation (8
). Since the concentrations of decitabine used here are well below those used in cancer treatment (31
), it seems unlikely that decitabine, as part of an anti-HIV therapy regimen, would introduce mutations into genomic DNA, given the presence of host DNA repair mechanisms. This model is supported by our data, which did not show an increase in C-to-G mutations, the mutations expected if decitabine were not excised from the viral DNA during integration. Instead, our data suggest that decitabine is removed after integration by the host DNA repair machinery and that the G-to-C mutations are due to the guanosines that replace decitabine in the minus strand viral DNA. Furthermore, decitabine was not genotoxic, as determined with the HGPRT assay by us (data not shown) and others (32
). Additionally, decitabine was not mutagenic to male Fisher rats that were treated with decitabine for 1 year (6
). Despite this evidence that decitabine is not mutagenic to the human genome, there was one report that decitabine was mutagenic in a transgenic mouse model (23
Although it is possible that the combination of decitabine and gemcitabine decreases infectivity by more than one mechanism, an additional antiviral activity would not negate the data presented, which show that completion of RT in the presence of decitabine and gemcitabine significantly alters the mutation spectrum. Although decitabine is known to affect the methylation of cellular genes, it is unlikely that this activity contributes to the decrease in infectivity shown here (5
). Similarly, there is no evidence that decitabine acts as a chain terminator to inhibit replication. In fact, while the combination of decitabine and gemcitabine modestly inhibited viral DNA synthesis, the decrease in RT products was not enough to account for the loss of infectivity. Since mutagens are not expected to act as chain terminators, no significant decrease in viral DNA would be expected from compounds acting as lethal mutagens. In fact, previous studies have demonstrated that even when mutagens eliminate infectivity, there are still detectable levels of viral RNA (13
). This is likely due to the production of defective viruses that are not infectious and can interfere with the infectivity of viruses that were not lethally mutagenized, a process known as lethal defection (13
The combination therapy we have identified in this study has many advantages, including that (i) it has a low effective concentration of each drug due to synergy, which decreases concerns of host toxicity; (ii) it uses a nucleoside analog that can be incorporated into the HIV genome but is unlikely to be incorporated into the host genome or, if incorporated, may be efficiently excised from the human genome; and (iii) both of the drugs used are already approved for human clinical use for conditions other than HIV infection, which should shorten the time and decrease the cost associated with drug development. Given these advantages, the combination of decitabine and gemcitabine may offer a novel treatment option for HIV-infected individuals. More importantly, this study reveals a novel treatment strategy of combining a nucleoside analog and a ribonucleotide reductase inhibitor to decrease infectivity through an increase in the HIV mutation rate.