Despite over 3 decades of intense research, an estimated 35.3 million people today are living with HIV-1 (Human Immunodeficiency Virus 1), the etiological agent of AIDS (Acquired Immunodeficiency Syndrome). The good news is that deaths are declining, in large part to combinations of highly active antiretroviral drugs. However, all of the current treatment options do not amount to a cure and a vaccine to prevent future infections is not yet available. With nearly 2 new infections for every 1 patient beginning an antiretroviral treatment regimen, the need is as great as ever to explore every avenue for new therapeutic interventions. One of these avenues is harnessing proteins endogenous to the very cells targeted for infection by HIV-1. Collectively referred to as host restriction factors, these innate immune proteins share a number of characteristics. First, they decrease virus replication. Second, expression of host restriction factors is often inducible and coupled to the immediate innate immune response to a foreign attack. Third, under the immense pressure of adapting to a pathogenic agent, host restriction factors often exhibit high degrees of rapid evolutionary change. Finally, if a restriction factor is potent enough, the virus will have evolved a counter-restriction mechanism. My thesis research has focused on the APOBEC3 family of host restriction factors that constitute an important arm of innate immunity. These enzymes, armed with the capacity to trigger cytosine to uracil mutagenesis in single-stranded DNA, function to defend the genome against attacks from foreign DNA elements including the retrovirus HIV-1. A more comprehensive understanding of the entire APOBEC3 family, its cellular functions, and how HIV-1 counteracts these activities with its accessory protein Vif, will all be essential before the design of therapeutic approaches incorporating endogenous APOBEC3 proteins in the fight against HIV/AIDS can be achieved. All 7 APOBEC3 proteins have been implicated in restricting HIV-1. The overall objective of my thesis research, detailed in the following chapters, was to define which of the 7 APOBEC3 proteins are capable of restricting the replication of HIV-1 in a T cell and which are potentially the source of the high levels of G-to-A mutagenesis observed in patient-derived HIV-1 sequences. To begin to narrow down which proteins are involved in vivo, we solved a long-standing problem in the APOBEC3 field. Quantifying expression levels had been impeded by the degree of homology between APOBEC3s, both at the nucleic acid and protein levels. My colleagues and I developed and characterized specific and efficient expression assays for each APOBEC3 family member. Using these assays we demonstrate that multiple APOBEC3s were expressed in relevant cell types and tissues including the major target of HIV-1, CD4+ T lymphocytes. To interrogate the contribution of individual APOBEC3s to HIV-1 restriction, we took a genetic approach and performed targeted deletion and knockdown experiments in a cell line that expresses multiple APOBEC3s. This approach allowed for a direct and definitive test of our hypothesis that multiple endogenous APOBEC3s restrict Vif-deficient HIV-1 replication. Based on HIV-1 replication kinetics and the levels of viral genome mutation, we concluded that 4 APOBEC3s are involved in HIV-1 restriction and that any future strategies employing the restrictive APOBEC3s will benefit from liberating all four proteins from Vif counteraction rather than any single one alone.Finally, to explore the consequences of genetic variation in the APOBEC3 locus on the HIV-1 restriction capacity of these proteins, we analyzed the expression levels and activity of the 7 haplotypes of the most genetically diverse APOBEC3, APOBEC3H. Through a series of primary cell experiments and HIV-1 spreading infections, we found that a subset of APOBEC3H haplotypes produce proteins that are stably expressed and capable of restricting naturally occurring HIV-1 variants that haven't evolved or have lost the capacity to neutralize this APOBEC3. The ramifications of this variability in a human restriction factor coinciding with diversity in HIV-1 variants able to counteract it will be an exciting area of future research. Overall, my research demonstrated that HIV-1 must contend with 4 APOBEC3 proteins to efficiently replicate in T cells. Expression of the APOBEC3s in human cells is widespread and inducible, and these host restriction factors combine to mutagenize the viral genome in the absence of HIV-1 Vif or, in the case of APOBEC3H, with Vif-proficient HIV-1 variants that are unable to mount an effective counter-defense. Consequently, unleashing all of these potent antiviral agents and allowing them to directly attack the virus' genetic code may lead to the first targeted innate immune therapy against HIV-1.