Background:  APOBEC3G (A3G), a member of the cytidine deaminase family, acts as an anti-HIV innate mechanism, which is circumvented by the HIV Vif protein. We previously found that the expression of endogenous A3G greatly differed among distinct normal DC subsets and that DC maturation induced a strong up-regulation of its expression. The endogenous A3G expression and its subcellular visualization in primary non-transfected CD4⁺ T cells, the main target for HIV infection and replication, remains unknown. In this study we have investigated this issue in normal resting and in activated proliferating CD4⁺T cells.

Methods:  Peripheral blood mononuclear cells (PBMC) from healthy subjects cultured for different periods of time with or without activation stimuli, phytohemagglutinin (PHA), the T-cell superantigen Staphylococcus enterotoxin A (SEA) and phorbol myristate acetate (PMA) in the presence or absence of interleukin-2 (IL-2). mRNA A3G expression was assessed by a real-time polymerase chain reaction (RT-PCR). Intracellular staining assessed by flow cytometry and confocal microscopy were used to assess A3G protein expression and its subcellular localization using our anti-A3G mouse monoclonal antibody, Apo-7, which detects a conformational epitope and also allows immunoprecipitation.

Results:  A 3-day culture of normal PBMC with PHA and SEA, but not with PMA, resulted in a great increase of A3G mRNA expression. Flow cytometry also showed a robust increase of A3G protein expression in CD4⁺ T cells after 3 days of activation with SEA and PHA. This increase was more prominent after a 7-day culture period in the presence of IL-2, and was also confirmed by immunoprecipitation. Confocal microscopy examination, apart from corroborating the A3G protein up-regulation in SEA- and PHA-activated CD4⁺ T cells compared with resting CD4⁺ T cells, demonstrated that an important proportion of A3G protein translocated to the nucleus, whereas it remained perinuclear in resting CD4⁺ T cells.

Conclusions:  In activated CD4⁺ T cells, the main cell target where HIV replicates, endogenous A3G protein undergoes a strong up-regulation and largely translocates from the cytoplasm to the nucleus. Whether the translocated A3G has anti-HIV activity remains to be determined. Experiments are being done to assess whether A3G is a nucleocytoplasmic shuttling protein, as has been demonstrated for APOBEC1 and activation-induced deaminase (AID).