Cytokine-mediated Regulation of APOBEC3G Expression and Complex Formation in Primary Immune Cells
Kimberly Stopak, J. David Gladstone Institutes
Biomedical and Clinical Sciences
Background: HumanAPOBEC3G (A3G),a deoxycytidine deaminase, is abroadly-acting antiretroviral factor. Given the antiviral effects ofA3G, cytokine-stimulatedT cells may upregulateA3G as a means to counter viral infection. Conversely, various cytokines such as IL-2, IL-4, IL-7, and IL-15 have been reported to convey permissivity to HIV infection in resting CD4 T cells. A3G can exist in two different forms, specifically an enzymatically-active low molecular mass form and an enzymatically inactive, high molecular mass A3G complex containing one or more RNAs. LMMA3G ischaracteristically found in resting CD4T cells where it functions as a potent post-entry restriction factor, while HMM A3G complexes are found in mitogen-activated CD4 T cells (see Chiu et al., Nature 435: 108, 2005).We have surveyed a range of cytokines to determine their effect on both A3G expression and A3G complex assembly.
Methods: Purified CD14- peripheral blood lymphocytes were stimulated individually with IL-2, IL-4, IL-6, IL-7, IL-9, IL15, TNFalpha, and IFNalpha, beta, and gamma; and the potential induction of A3G mRNA and protein was assessed. Interferonalpha-treated macrophages and mature versus immature dendritic cells were also assessed for potential changes in the expression of A3G. In parallel, the potential involvement of select signaling pathways in the observed response was tested by adding inhibitors specific for the Jak/Stat and MAP kinase pathways. Finally, using fast protein liquid chromatography, we assessed whetherA3G was present in the LMM or HMM form after stimulation of purified CD4+ T cells with these cytokines.
Results: A3G expression at the protein level in PBLs was induced by IL-2 and IL-15, and, to a lesser extent, IL-7. No response was observed with IL-4, IL-6, IL-9, TNFalpha or the alpha, beta, or gamma isoforms of IFN. This increase in A3G protein was mirrored by upregulation of A3G mRNA detected by quantitative real time PCR. Inhibition of JAK or MAPK activation sharply impaired the induction of A3G in response to IL-2, IL-7, and IL-15. Dendritic cell maturation and interferon-treatment of macrophages was also associated with a marked increase in the expression of A3G. When A3G complex formation was assessed, IL-2, IL-7 and IL-15 each promoted HMM A3G complex assembly in purified CD4 T cells.
Conclusion: We conclude that IL-2, IL-7, or IL-15 stimulation of resting CD4 T cells leads to transcriptional activation of theA3G gene resulting in increasedA3G mRNA and protein ex-pression. However, the potent post entry restricting activity of the LMM form of A3G is forfeited because the induced A3G protein is recruited into inactive HMM A3G RNA-protein complexes. These effects may explain why these cytokines cause resting CD4 T cells to become permissive to HIV-1 infection.