CHARACTERIZATION OF APOBEC3 REPERTOIRES AND EFFECTS ON RETROVIRAL REPLICATION

APOBEC3 (A3) enzymes are a family of intrinsic retroviral restriction factors that are coordinately expressed in CD4+ T-cells and function to restrict retroviral replication. In the case of HIV-1, this primarily occurs in the absence of the HIV-1 protein, viral infectivity factor (Vif). Vif induces the polyubiquitination and degradation of A3 enzymes using the host proteasome pathway. The A3 enzyme family are single stranded DNA deaminases, capable of deaminating cytosine to form promutagenic uracil on single-stranded DNA substrates. In humans there are seven paralogs that comprise the A3 family, including: A3A, A3B, A3C, A3D, A3F, A3G, and A3H. However, only four paralogs have been associated with inhibition of retroviral replication in a large majority of the human populations, including: A3D, A3F, A3G and A3H. These four key enzymes have well-characterized repertoires of single nucleotide polymorphisms (SNPs) that affect both cellular stability and deaminase activity. Each repertoire of polymorphisms is variable in human populations and is influenced by human ancestry. Here we have undertaken a Saskatchewan-based mixed population study examining human genotypes for common APOBEC3H (A3H), APOBEC3F (A3F) and APOBEC3G (A3G) SNPs and how combinatory SNP variations affect HIV-1 replication. Using Sanger-sequencing based genotyping we were able to isolate dominant polymorphisms in our population. We found that the dominant genotypes in our population were heterozygous for A3F 231V and 231I SNPs, homozygous for the A3G 186H SNP, and predominantly inactive A3H SNP profiles. We tested the dominant A3F and A3G polymorphisms for cellular stability, viral packaging and restriction capacity, and found that the A3F 231V SNP provided the most robust restriction response when co-expressed with both A3G and A3F 231I. We also show that A3F 231V can hetero-oligomerize with both A3G and A3F 231I polymorphic variants, resulting in greater cellular stabilization and steady-state cellular expression in the presence and absence of HIV-1 Vif. The observed rise in cellular stability and virion packaging when A3F 231V was co-expressed with A3F 231 and A3G had a positive correlation with HIV-1 replication restriction efficiency. The various states of oligomerization between co-expressed A3 enzymes demonstrates that whole genotype analysis of A3 repertoires is essential in accurately understanding host-pathogen interactions on a population level