T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.: APOBEC3G (A3G) is an antiviral enzyme that is expressed in human T cells and macrophages, which are the cell types infected by HIV. Early in the HIV life cycle, the HIV RNA genome is reverse transcribed into DNA. A3G can modify this DNA enzymatically, leading to high rates of mutation such that the virus can no longer replicate. To date, most studies of A3G's enzymatic activity have utilized cell lines (293T and HeLa) that can be transfected to express A3G but do not express it endogenously. A report of unexpectedly low levels of mutation in viral DNA from HIV-infected human T cells led us to investigate regulation of A3G enzymatic activity in T cells. We developed a high-throughput assay to compare the enzymatic activity of endogenous A3G in T cells versus transfected (exogenous) A3G. Surprisingly, enzymatic activity of A3G from human T cell lines and primary T cells was very low relative to A3G from transfected cells, even when corrected for A3G protein amount. Moreover, T cell lysates inhibited enzymatic activity of exogenously expressed A3G. These data suggest that enzymatic activity of endogenous A3G in human T cells is inhibited by an uncharacterized mechanism that may protect the host from this DNA mutator and could have important implications for A3G antiviral activity in vivo.