HIV-1 Tat is known to influence several intracellular functions including its ability to activate long terminal repeat (LTR) promoter-mediated transactivation and cause apoptosis [1–3]. Although a number of studies have been performed with subtype B gene products, relatively little information is available for subtype C, which is responsible for causing more than 50% infections worldwide, including India where it is the major subtype. In the present study, we constructed several recombinant clones of Tat-B (derived from pNL4-3)  and Tat-C (derived from an Indian isolate which is ∼95% similar to the consensus subtype C based on its amino acid sequence  (clone 93IN905, GenBank Accession no. AF067158)  with respect to the two exons for fine functional domain analysis. It is worth noting that most subtype C isolates possess QGD compared with RGD in the second exon of Tat gene in the same position (78–80 amino acids) (a known cell adhesion motif), which is associated with integrin-mediated signaling and cell adhesion, etc., besides other changes throughout the gene. We reasoned that these changes might modulate its ability to transactivate LTR promoters and apoptosis. Therefore, we made constructs of subtypes B and C Tat that consisted of either the RGD or QGD motif and also swapped the first and second exons of the Tat gene. Precise gene fusion technology was used to generate such chimeric constructs as described by one of us earlier  and confirmed by sequencing. The various constructs made are indicated at the bottom of Fig. 1a with Tat-B and Tat-C with RGD/QGD domains. An internal reporter gene control (pSV-β-gal; Promega, Madison, Wisconsin, USA) was always included to ensure uniform transfection efficiency.
The various constructs were tested for their ability to activate either LTR-B-mediated or LTR-C-mediated transactivation in human Jurkat T cells as described earlier [8,9] and the results from three independent experiments (mean ± SD) are shown in Fig. 1. Lane 1 shows the background activity from cells only with LTR-B reporter plasmid DNA; an approximately 17-fold increase with wild-type Tat-B (lane 2); but an additional almost three-fold increase with Tat-B construct, which possessed QGD motif in place of RGD (hereafter called Mt-1 construct) (lane 3). The wild-type subtype C construct showed similar activity as obtained with wild-type B construct (compare lanes 2 and 4). The Tat construct possessing wild-type subtype C exon but the RGD motif (found in the second exon of subtype-B, hereafter referred to as Mt-2), showed significant reduction compared with wild-type B or wild-type C constructs. Lanes 6–10 represent the same experiment that was repeated with LTR-C-Luciferase DNA, which essentially gave the same pattern of promoter activation. An exactly similar pattern was observed when the same experiment was carried out on human 293 cells (data not shown).
We next evaluated the extent of apoptosis in PMA-stimulated THP-1cells (transformed human macrophages) caused by various wild-type and chimeric (B/C) Tat constructs (indicated at the top of each panel) including the possible role of RGD/QGD motifs only and the representative results from three independent experiments are shown in Fig. 1b. As expected, control cells showed almost 11% apoptosis (panel I); construct with both wild-type Tat-B exons, showed approximately 29% apoptosis (panel II); with Mt-1 construct, it remained the same as with wild-type B (panel III); wild-type Tat-C showed approximately 14% (panel V) apoptosis, which is about 15% less than what was observed with wild-type Tat-B in panel II. Interestingly, in the construct where first exon of Tat-B was fused with second exon of Tat-C, a much reduced cell death was observed (panel IV). The most interesting results were obtained with Mt-2 construct, which showed approximately 10% more cell death (panel VI) when compared with the wild-type Tat-C. A chimeric Tat construct with first exon fused with second exon of Tat-B (both wild type) showed intermediate levels of cell death (panel VII).
The following important conclusions can be drawn from this study. Replacing RGD motif with QGD alone in the second exon of Tat-B resulted in 2.5–3-fold additional increase in LTR promoter activity. Furthermore, when QGD motif in the second exon of Tat-C is replaced with RGD motif, a significant reduction in LTR-mediated promoter activation was observed. We conclude that RGD motif negatively regulates the Tat-mediated LTR-promoter activation in subtype B and C Tat. Wild-type subtype B Tat caused more cell death than wild-type subtype C. When the QGD motif present in the second exon of Tat-C was replaced with RGD motif of Tat-B, increased apoptosis was observed. Thus, RGD motif alone contributes substantially to Tat-C-mediated apoptosis. These findings are important with respect to further understanding the molecular basis of pathogenesis.
This work was supported by a grant from Department of Biotechnology, Government of India, to NII, ND, India and to the corresponding author (A.C.B.). Several HIV-1-related research materials (pNL4-3 DNA, LTR-B and C-Luciferase constructs, THP-1 cells, etc) were obtained from AIDS Research and Reference Reagent Program of NIH, MD, USA.
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