Unconventional measures are needed in an effort to outpace the HIV/AIDS pan- demic. Even with access to the six classes of FDA-approved antiretrovirals, adverse drug effects and evolution of drug resistance still pose obstacles, and represent issues that will inevitably arise in the developing world. Along with the absence of a proven vaccine strategy, these shortcomings necessitate the continual search for new therapeutic targets. One such novel approach is to target HIV-1’s low copying fidelity with a deliberate in- crease in viral mutational load using exogenous small molecule mutagens. However, in- asmuch as this concept of viral lethal mutagenesis has gained notoriety, it is also essential to understand fundamental enzymatic components (i.e., reverse transcriptase) that con- tribute to variation. The goal of this dissertation was to advance basic models to better understand the causes and consequences of HIV-1 variation. The components that influence variation within an HIV-1 population structure are critical to predict the emergence and direction of viral evolution (e.g., drug resistance). For these studies, the relationship between viral fitness and mutation rate was investigat-ed. A panel of 10 reverse transcriptase mutants – most having drug resistance pheno- types – were analyzed for their effects based on these two biological properties. Muta- tion rate differences were measured using single-cycle vector assays, while fitness differ- ences were identified using ex vivo head-to-head competition assays. As anticipated, vi- ral mutants possessing either higher or lower mutation rate had a corresponding loss in fitness. These observations provided the first description of an interrelationship between HIV-1 fitness and mutation rate and support the conclusion that mutator and antimutator phenotypes correlate with reduced viral fitness. A second focus of this dissertation was directed at studying novel mechanisms by which viral mutagens diminish HIV-1 infectivity. I have detailed the antiviral mecha- nisms of the ribonucleoside analog 5-azacytidine. It was demonstrated that the primary antiviral activity of 5-azacytidine can be attributed to its effect on the early phase of HIV- 1 replication mediated by reverse transcriptase. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants. Sequencing analysis showed enrichment in guanosine-to-cytidine (G-to-C) transversion mutations. These re- sults indicated that 5-azacytidine was incorporated into viral DNA following its 2’-OH reduction to 5-aza-2’-deoxycytidine. Incorporation into the viral DNA led to an increase in mutant frequency, which is consistent with lethal mutagenesis. Lastly, studies were directed at understanding concomitant exposure of two unre- lated mutagenic agents. Because the APOBEC3 proteins’ restrictive nature associated with cytosine deamination, I wanted to investigate its interplay with the small molecule cytosine analog, 5-azacytosine. Reduced viral infectivity and increased viral mutagenesis were observed with both the viral mutagen 5-azacytosine (i.e., G-to-C mutations) and the host restriction factor APOBEC3G (i.e., guanosine-to-adenosine (G-to-A) mutations); however, when combined, they had complex interactions. Nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-azacytosine and APOBEC3G result- ed in an increase in G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. Al- so, APOBEC3G catalytic activity was required for the diminution in G-to-C mutagenesis. These findings provided the first demonstration for potentiation of the mutagenic effect of a cytosine analog by APOBEC3G expression, resulting in concomitant HIV-1 lethal mutagenesis. In summary, the studies conducted in this dissertation 1) provide the first direct experimental evidence of an interrelationship between HIV-1 fitness and mutation rate, 2) demonstrate that the primary antiviral mechanism of 5-azacytidine can be attributed to its ability to increase the HIV-1 mutation frequency through viral DNA incorporation during reverse transcription, and 3) provide the first demonstration for potentiation of the muta- genic effect of a cytosine analog (i.e., 5-azacytosine) by APOBEC3G expression, result- ing in concomitant HIV-1 lethal mutagenesis.