Lethal mutagenesis as a therapeutic strategy has gained momentum, especially after ribavirin and KP-1212 gained clinical use 9; 11
. Most of the exploratory HIV-1 mutagens target C analogs including the triazole base, 5-AZC. Since the APOBEC3 protein subfamily also targets C bases for deamination, these mutagens may exert redundant mechanisms when combined. This would suggest that compounds targeting C bases could be less effective mutagens in the presence of A3G. Alternatively, incorporation of 5-AZC could have secondary effects by altering A3G substrate specificity or target site architecture. In this study, we examined concomitant exposure of HIV-1 to 5-AZC and A3G. The ability of A3G to induce G-to-A hypermutagenesis provides an example of an evolutionary conserved mechanism that eliminates HIV-1 infectivity by lethal mutagenesis. Editing of retroviral genomes by A3G in the face of concomitant mutagen exposure has not been previously explored.
5-AZC is a first-in-class hypomethylating agent as substitution of N-5 can no longer be methylated 40
. The ribonucleoside 5-AZC undergoes anabolic metabolism to the corresponding triphosphate, but a fraction is converted to the nucleotide triphosphate, 5-aza-2′-deoxycytidine (5-AZdC), and is substituted for dCTP in DNA 41; 42
. Because aza-pyrimidines are chemically unstable, ring-opened intermediates may have the potential for non-Watson-Crick base pairing, leading to base mispairing 43
. In fact, 5-AZC has been implicated in several studies as a mutagen, specifically introducing a rare GC-to-CG transversion type 7; 44; 45; 46
. Experimentation with an HIV-1 vector system found that 5-AZC increased the preponderance of only G-to-C mutations, suggesting that the mutagenic product of azacytosine is able to template for C:C mispairings when incorporated during minus-strand DNA synthesis 7
Intriguingly, the majority of retroviral minus-strand DNA is also transiently single-stranded during replication, and ssDNA is the preferential substrate of the APOBEC3 subfamily 14
. During HIV-1 reverse transcription, A3G gains access to ssDNA, targeting cytosine residues for deamination to pre-mutagenic lesions 47
. These kinetics, of A3G and 5-AZC-based mutagenesis, suggest that access to minus-strand ssDNA by A3G is subsequent to RT incorporation of 5-AZdC during minus-strand DNA synthesis.
The interaction between A3G and the non-canonical 5-azacytosine base could result in two possible obvious outcomes: 1) A3G is unable to engage 5-azacytosine or its ring-opened decomposition products. This would be observed as a decrease in G-to-A mutations with no difference in the number of G-to-C mutations; 5-azacytosine would effectively antagonize A3G deaminase activity. 2) A3G is able to catalyze deamination of 5-azacytosine to 5-azauracil and this uracil analog, results in increased G-to-A mutations at the expense of G-to-C mutation types. However, other mechanisms cannot be excluded at this time, such as: the ability of 5-azacytosine (or its ring-opened products) to alter local DNA secondary structure and subsequent ssDNA availability, or the influence of 5-azacytosine on A3G processivity or substrate specificity due to close-proximity base interactions, as suggested by Rausch et al. 48
In order to better understand mutation type-specific differences of concomitant mutagen exposure, proviral DNAs were sequenced. The G-to-A mutational load (i.e., # of G-to-A mutations/proviral clone) was increased up to 18% in virus exposed to both 5-AZC and A3G, as compared to A3G exposure alone. This effect was at the expense of a 36% decrease to the 5-AZC-induced mutational load (i.e., # of G-to-C mutations/sequence). These results suggest a model whereby 5-AZC incorporation into retroviral DNA causes complex interactions with A3G, to inversely shift the G-to-A and G-to-C mutational loads. Furthermore, the E259Q catalytically inactive A3G, in conjunction with 5-AZC, showed no difference in G-to-C transversions compared to drug alone, suggesting a requirement for fully functional A3G in order to observe the increase of G-to-A mutations at the expense of G-to-C mutations.
One potential concern regarding the sequencing data is biased amplification due to the oligonucletides selected. Particular attention to primer design helped ensure universal and unbiased PCR. For example, the location of each oligonucleotide was carefully adjusted such that no di-GG nucleotide motif was positioned in the forward primer (and no di-CC nucleotide motif in the reverse primer) in order to eliminate the potential for mis-annealing due to the creation of A3G signatures. Moreover, no more than two G nucleotides were positioned at the forward primer regions (no more than two C nucleotides in the reverse primer region), while these potentially mutable bases were substituted with the degenerate S (50% G and 50% C), to exclude 5-AZC biased amplification. These primers amplified heavily mutated sequences in each treatment group (23 mut/720 bp), indicating that the sequenced DNAs were not biased against any mutation type.
Presently, concomitant use of viral mutagens and chain terminators shows efficacy against RNA viruses 49; 50; 51; 52; 53; 54
. For example, Perales et al. demonstrated that in foot-and-mouth disease virus, sequential treatment with a traditional antiviral inhibitor followed by the viral mutagen ribavirin was much more effective in extinguishing picornavirus replication than these compounds used together, or either one alone 53
. Future progress of mutagen utilization hinges on understanding the mutational constraints within RNA virus population structure. For example, sub-restrictive editing of retroviral genomes by APOBEC3 proteins has the potential to negatively influence therapeutic intervention, by rapid generation of drug-resistant mutants 55; 56
. Furthermore, quasispecies theory predicts that populations will evolve more robust genomes, termed survival of the flattest, in the face of increased mutational load 57; 58
. Alternative adaptive strategies include selection of anti-mutator viral polymerases 59; 60; 61
or enhanced discrimination between correct and mutagenic nucleotides 62
. Evolution of drug resistance, or increased robustness, of mutagens can threaten such therapeutic approaches; yet, discovery of novel viral mutagens, as well as optimal applications, may lead to alternative therapeutic strategies.
Understanding molecular details of potential mutagens as well as sequence space limitations of specific viral pathogens may provide a rationale for tailored therapeutic intervention rather than lengthy small molecule screening. Generally, HIV-1 viral mutagens are stealth nucleosides directly utilized by RT during replication to induce site-specific mutations. These compounds are referred to as universal bases because they can mispair with more than one of the canonical Watson-Crick base pairs. For instance, the viral mutagen KP1212, can base pair with either G or A due to a tautormeric shift in the pyrimidine base. 5-AZC, a close derivative to KP1212, induces a unique G-to-C transversion mutational pattern during HIV-1 replication because of its ability to mispair with C bases 7; 43; 44
. Even by understanding specific molecular details, viral mutagens still lack a reliable framework to help predict successful treatment outcomes. Many parameters involved in understanding virus population dynamics and genetic diversity are not fully understood, including: sequence space, mutational robustness, effective population size, as well as the natural fitness landscape. Similarly, since nucleotide base composition among viral genera is known to be quite distinct, purposeful alterations to the mutational bias of a particular virus may pose a greater defect to viral fitness. For example, since HIV-1, like other lentiviruses, has an unusually A-rich genome 63
, it is not clear if mutagens that cause more N-to-A mutations (versus A-to-N) are more detrimental to viral fitness.
In summary, we describe concomitant HIV-1 mutagenesis using two unrelated classes of viral mutagens. Our findings indicate a combined, yet intricate, interaction between the 5-AZC and A3G. The combined antiviral effect observed indicated that A3G potentiated the mutagenic effect of 5-AZC. Sequencing analysis revealed that the combined mutagenic effect resulted in an increase in the frequency of G-to-A mutations at the expense of G-to-C mutations, suggesting a complex interaction between A3G and 5-AZC upon incorporation into viral DNA. Future studies will provide greater details into the molecular mechanisms involved in concomitant HIV-1 lethal mutagenesis by A3G and 5-AZC.