Telomerase activity remains unchanged alter HIV-1 Tat expression in GHOST-cells: comparative analysis between HIV-1 subtype B and BF isolates

REYNOSO RP; VIGNOLES M; TURK GJ; MINCES LR; SARACCO M; SALOMON H; QUARLERI JF

Rio de Janeiro, Brasil

Congreso; 3rd IAS Conference on HIV Pathogenesis and Treatment.; 2005

International AIDS Society

Introduction: HIV-1 has been associated to accelerated cellular senescence. The objective of the present study was to analyze, in vitro, the role of Tat protein as a viral factor potentially involved on that phenomena by down-regulating telomerase activity.HIV-1 has been associated to accelerated cellular senescence. The objective of the present study was to analyze, in vitro, the role of Tat protein as a viral factor potentially involved on that phenomena by down-regulating telomerase activity. Methods: A GHOST(3) CXCR4 cell line was transfected with pTARGET/TatB, pTARGET/TatBF expression vectors. These vectors contained full length Tat coding sequences from pNL4-3 (subtype B) and from a CRF12_BF isolate. Mock vectors and untransfected cells were used as negative controls.  Forty-eight hours after transfection cells were harvested and split for telomerase activity quantification (Roche Diagnostics, Germany) and Tat expression evaluation. The latter was evaluated by GFP expression (transactivated by tat) in GHOST-cells: cells were fixed and analyzed by flow cytometry. RTCN (ratio to cell negative) was calculated as the proportion between percentage of fluorescent-positive cells and the mean fluorescence intensity. Two independent experiments were carried out in triplicate. Statistic analysis was done using a parametric test (Student´s t-test).A GHOST(3) CXCR4 cell line was transfected with pTARGET/TatB, pTARGET/TatBF expression vectors. These vectors contained full length Tat coding sequences from pNL4-3 (subtype B) and from a CRF12_BF isolate. Mock vectors and untransfected cells were used as negative controls.  Forty-eight hours after transfection cells were harvested and split for telomerase activity quantification (Roche Diagnostics, Germany) and Tat expression evaluation. The latter was evaluated by GFP expression (transactivated by tat) in GHOST-cells: cells were fixed and analyzed by flow cytometry. RTCN (ratio to cell negative) was calculated as the proportion between percentage of fluorescent-positive cells and the mean fluorescence intensity. Two independent experiments were carried out in triplicate. Statistic analysis was done using a parametric test (Student´s t-test). Results: The average RTCN values after 48 hours of transfection were 510 and 398 for cultures transfected with pTARGET/TatB and pTARGET/TatBF, respectively. Average telomerase activity (expressed as Relative Telomerase Activity) showed no significant variations (p<0.05) when comparing mock cells vs. TatB constructions, as well as when comparing mock cells vs. TatBF constructions.The average RTCN values after 48 hours of transfection were 510 and 398 for cultures transfected with pTARGET/TatB and pTARGET/TatBF, respectively. Average telomerase activity (expressed as Relative Telomerase Activity) showed no significant variations (p<0.05) when comparing mock cells vs. TatB constructions, as well as when comparing mock cells vs. TatBF constructions. Conclusions: HIV-Tat produced on GHOST transfected cells could not be essential on telomerase activity down-regulation. Likewise, no differences were observed between B and BF-based constructions. It has been previously reported that extracellular Tat down-regulates telomerase activity; our observations could imply that Tat action might be ascribed to a bystander pathogenic effect promoting ageing on infected cells.HIV-Tat produced on GHOST transfected cells could not be essential on telomerase activity down-regulation. Likewise, no differences were observed between B and BF-based constructions. It has been previously reported that extracellular Tat down-regulates telomerase activity; our observations could imply that Tat action might be ascribed to a bystander pathogenic effect promoting ageing on infected cells.