PMC:1920263 / 23089-27540
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1920263","sourcedb":"PMC","sourceid":"1920263","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1920263","text":"The A3G promoter is not inducible in T cells\nAccording to the current knowledge, APOBEC3G plays a role in the innate defence against pathogens like HIV-1. The latter has evolved a mechanism to counteract A3G activity by expressing the regulatory protein Vif (4). Vif induces proteasomal degradation of A3G and additionally inhibits A3G activity by further mechanisms (8, 37–43). To investigate whether the overexpression of HIV-1 proteins also influences A3G promoter activity, we either expressed HIV-1 Vif or the HIV-1 Tat protein, the latter has been shown to activate different viral and cellular promoters (44,45). In addition, increasing amounts of the plasmid pNL4-3, containing the full-length genome of HIV-1, were transfected. These constructs or the empty vector pcDNA3.1 were cotransfected with the 1025 bp construct into A3.01 T cells. Luciferase assays showed that the transcriptional activity of the A3G promoter was not significantly altered in the presence of Vif, Tat or HIV-1NL4-3 (Figure 4A), suggesting that HIV-1 is not modulating A3G expression on the transcriptional level.\nFigure 4. A3G promoter activities after coexpression of HIV-1 proteins or treatment with TPA or interferons. (A) A3.01 T cells were cotransfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter and 1 µg of Vif expression plasmid, 1 µg Tat expression plasmid or increasing amounts of HIV-1NL4-3 (0.1, 0.5 and 1 µg). After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (B) A3.01 T cells were transiently transfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter or with empty vector. Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 20 ng/ml TPA. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities and the values for the empty vectors (untreated and TPA-stimulated) were set as 1. (C) A3.01 T cells were transiently transfected with the A3G promoter constructs or with the interferon-responsive reporter plasmid pGL2-CVX (GAS). Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 30 ng/ml IFN-α or IFN-γ. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (D) HepG2 cells were used for transfection. The experiment was performed as described in (C). Mean values (±SD) of representative experiments performed in triplicate are shown.\nIt has been shown that the amount of A3G mRNA in T cells is increased in response to mitogenic stimulation with phorbol ester (46–48). In addition, interferons have been described to upregulate A3G expression in hepatocytes and macrophages (46,48). To investigate whether these stimuli interfere with A3G promoter activity in T cells, we transfected A3.01 T cells with the 1025 bp promoter or the empty vector pGL3-Basic (vector) and treated the cells with phorbol ester (TPA). The luciferase assay showed that the ∼15-fold increased transcriptional activity of the 1025 bp promoter relative to the empty vector was not further enhanced by TPA treatment (Figure 4B), although the functional activity of TPA was confirmed by induction of the SV40 promoter-containing reporter plasmid pGL3-Control (data not shown). Similarly, treatment of A3.01 T cells transfected with the A3G promoter deletion constructs with IFN-α or IFN-γ showed no effect (Figure 4C). Interestingly, a control plasmid (pGL2-CVX) containing two IFN-responsive GAS (gamma activated sequence) elements upstream of the luciferase reporter gene was only induced by IFN-α in these cells (Figure 4C). Since two reports describe A3G upregulation by interferons in hepatocytes (47,48), we additionally performed the experiment in the hepatic cell line HepG2. In line with these publications, we observed an induction of the A3G promoter by approximately 2-fold after IFN-α or IFN-γ stimulation (Figure 4D) with IFN-γ being slightly more potent. For both interferon types, induction of A3G promoter activity was observed for all deletion constructs except for the 60 bp fragment, indicating that the responsible region is located within the 60 nt present in the 120 bp, but not in the 60 bp fragment.","divisions":[{"label":"Title","span":{"begin":0,"end":44}},{"label":"Figure 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