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H. Hu; Expression of HIV–1 Tat in ARPE–19 Cells and Its Influence on the Function of Different Transporters . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1039.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: HIV–1 infection is associated with retinal complications, known as non–infectious AIDS retinopathy (NIAR), even in the absence of concurrent infections with other opportunistic viruses. HIV–1 genome codes for Tat, a protein essential for viral replication in host cells. Tat is found in the circulation of HIV–1–infected persons and is known to have a variety of biologic effects on mammalian cells. The Tat gene consists of two exons and the protein interacts with the long terminal repeat (LTR) region of HIV–1 genome. The biologic effects of Tat on various retinal cell types are at least partly responsible for the pathogenesis of NIAR. Here we investigated the influence of Tat on the transport function of RPE. Methods: We generated stable ARPE–19 cell lines that express either the 72–amino acid–long Tat from the first exon (Tat–1) or the 101–amino acid–long Tat from both exons (Tat–2). The expression of the Tat was confirmed by RT–PCR and immunofluorescence. The function of Tat was confirmed using an enhanced green fluorescence protein (EGFP)–linked reporter construct containing the HIV–1 LTR. The influence of Tat on transport function was assessed by monitoring the activity of four transporters: glutamate–cystine exchanger (x–c), excitatory amino acid transporter (EAAT), opioid peptide transporter, and creatine transporter. Results: RT–PCR analysis established that Tat–1 and Tat–2 cells expressed the 1–exon Tat and the 2–exon Tat, respectively. Immunofluorescence confirmed the expression of the Tat protein. Transient transfection of Tat–1 and Tat–2 cells with the HIV–1 LTR–EGFP reporter construct showed that both the short and long forms of Tat were able to interact with HIV–1 LTR. Functional analysis of various transporters showed that the expression of Tat increased the activity of x–c, opioid peptide transporter, and creatine transporter and decreased the activity of EAAT. These effects were comparable in Tat–1 cells and Tat–2 cells. Conclusions: Expression of Tat in ARPE–19 cells has marked effects on the transport function of these cells. The effects of Tat are comparable irrespective of whether the Tat protein is coded by only the first exon or by both exons.
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