Measurement of the range of HIV-LTR transactivating activity of HIV in vitro

Tat is known to act both in a 'cell autonomous' and in 'cell non-autonomous' manner, i.e. Tat can function as a powerful transcription factor both inside the cell in which it is expressed and also in cells not expressing Tat. However, the distance at which Tat can act by the cell non-autonomous mechanism is not yet known. To estimate the range of Tat action and to understand the mechanism underlying its exocrine action, we used HeLa cells stably expressing chloramphenicol-acetyltransferase (CAT) as a reporter gene under the control of the HIV-LTR. These cells were transiently co-transfected by the Kalashnikov bombardment technique with a plasmid carrying the beta-galactosidase reporter gene under the control of the HIV-LTR together with a plasmid expressing the Tat gene. The cells were then analyzed for expression of both CAT (LTR-transactivation marker) and beta-galactosidase (Tat expression marker). The results indicate that Tat can act by inducing the expression of LTR-CAT not only in the Tat producing cells, but also in neighboring cells up to 6-10 cells distance from the producer cell. Furthermore, we demonstrate that the Tat-RDC motif is essential for this paracrine action of Tat, since a Tat protein carrying a deletion of the RDG sequence was unable to activate either adjacent or distant cells. These results suggest the presence of different mechanisms through which exocrine Tat can exert its action.