Abstract
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.
Highlights
Viral infection represents a common threat faced by most cells, and different cell types have evolved unique strategies for defending against viral pathogens
APOBEC3G (A3G) is an antiviral enzyme that is expressed in human T cells and macrophages, which are the cell types infected by HIV
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
Summary
Viral infection represents a common threat faced by most cells, and different cell types have evolved unique strategies for defending against viral pathogens One such strategy involves the deoxycytidine deaminase APOBEC3G (A3G), an intrinsic defense mechanism specific to primates. The presence of multiple deoxyuridines (dUs) in the minus strand may prevent accumulation of reverse transcripts, either by triggering degradation by cellular DNA repair machinery [11,12,13] or by impairing synthesis [14,15]. In both cases, dC-to-dU deamination was initially thought to be critical to the antiviral effects of packaged A3G. Subsequent studies have demonstrated that packaged A3G mutants can have antiviral effects even when they lack deaminase activity [16,17,18,19]
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