Regulation of APOBEC3G enzymatic activity in HIV-infected primary human T cells

University Of Washington, Seattle WA

Investigators

Abstract

The human cytidine deaminases APOBEC3G (A3G) are antiviral proteins that cause DNA hypermutation in HIV-1 DNA by deaminating cytidines. A3G-mediated hypermutation of HIV DNA correlates with lower viral loads, emphasizing its importance in vivo, but unfortunately hypermutated proviral sequences are found in only 10-20% of patients. Thus, interventions to increase deamination of HIV in vivo could be useful therapeutically. However, little is known about the regulation of A3G enzymatic activity in primary T cells, which express A3G endogenously and are infected by HIV in vivo. Recently we established a high-throughput, non-radioactive assay for measuring A3G enzymatic activity. Using this assay we demonstrated that deaminase activity of endogenously expressed A3G in resting and fully activated primary human T cells is inhibited, suggesting that A3G is negatively regulated in primary T cells. Our preliminary data demonstrate that in primary T cells, A3G deaminase activity is regulated by signaling pathways raising the possibility that signaling pathways can influence the amount of HI hypermutation that occurs during infection. Here we propose to use our deaminase activity assay to understand the regulation of APOBEC3 (A3) proteins in primary T cells and how this regulation affects HIV infection. Specifically, we propose to 1) determine which signaling pathways activate A3G in primary T cells; 2) determine if phosphorylation state or association with known A3G interacting proteins correlates with A3G activation; 3) examine A3G enzymatic activity and hypermutation caused by A3G at different times during the HIV life cycle; and 4) understand the regulation of weakly active A3 proteins in primary cells that may promote HIV evolution. Thus, the studies proposed here will delineate how signaling pathways regulate endogenous deaminase activity of A3G in primary human T cells, and determine how this regulation influences generation of mutations in the HIV genome.