The Dynamic Interplay Between Lentiviral Vif and Human APOBEC3 Proteins

Abstract

Four members of the APOBEC3 (A3) family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with its Vif protein, which nucleates a ubiquitin ligase complex that directly binds A3 enzymes and targets them for proteasomal degradation. My thesis research aims at understanding the dynamic interplay between lentiviral Vif and human A3 enzymes, from the molecular determinants of this interaction to its implications in HIV-1 transmission within a large patient cohort. This has been addressed through three separate studies. The primate A3 repertoires show considerable variations likely due to positive selection during evolution. Lentiviral Vif proteins have rapidly evolved to counteract this immune pressure, resulting in present-day host-pathogen interactions that are largely species specific. However, a simian immunodeficiency virus (SIV) Vif exhibits cross-species degradation capability against multiple human A3 enzymes. We used mutagenesis coupled with functional assays to determine the residues involved in the interaction between the SIV Vif and A3B, and demonstrated that it resembles the HIV-1 Vif-human A3G interaction. This may be a molecular remnant of an ancestral Vif activity or result of molecular mimicry between human A3B and A3G. A3H is unique among family members by dimerizing through cellular and viral duplex RNA species. RNA binding is required for proper localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles, and ultimately for effective virus restriction activity. To investigate the role of RNA in HIV-1 Vif-mediated degradation of A3H, we used structural and cell biology approaches to study RNA binding mutants and their sensitivity to Vif-mediated degradation. We found that RNA is not strictly required for Vif-mediated degradation of A3H and that RNA and Vif bind the enzyme on largely distinct surfaces, but the degradation process may be affected by changes in subcellular localization/mobility and/or differences in the constellation of A3H interaction partners. In humans, A3H is the most polymorphic member of the family and includes seven haplotypes with three encoding for stable proteins and the rest unstable. Stable A3H proteins contribute to HIV-1 restriction and can only be counteracted by fully functional Vif variants (dictated by amino acids at key positions). We tested the hypothesis that stable A3H enzymes provide a transmission barrier to HIV-1 isolates harboring less-than-fully functional Vif alleles. We have determined the A3H and viral Vif genotypes of a large cohort of African HIV-1 serodiscordant couples and have shown stable A3H is unlikely to be a general protective factor in HIV-1 acquisition. However, stable A3H enzymes may still serve positive roles in slowing virus spread and disease progression. Overall, my thesis research contributes to the growing knowledge of the A3-Vif interaction, particularly interactions between the Vif protein of pandemic HIV-1 and the contemporary restriction factor A3H. These studies will help guide future efforts to disrupt this interaction as an antiviral therapy.