INHIBITION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT-DEPENDENT ACTIVATION OF TRANSLATION IN XENOPUS-OOCYTES BY THE BENZODIAZEPINE RO24-7429 REQUIRES TRANSACTIVATION RESPONSE ELEMENT LOOP SEQUENCES

Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiaza pin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1, 4] diazepin-2-yl]-methylamine (Ro24-7429), inhibit human immunode ficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR )-containing RNA is activated by Tat. Both compounds specifically bloc ked activation of translation in a dose-dependent fashion, with Ro24-7 429 showing the greater potency. In the Xenopus oocyte system, as in m ammalian cells, mutation of the TAR loop sequences abolishes Tat actio n. However, it is possible to obtain TAR-specific, Tat-dependent activ ation of a target RNA with a mutation in the loop provided that this t arget is in large excess. This result has been interpreted as indicati ng that a negative factor has been titrated (M. Braddock, R. Powell, A . D. Blanchard, A. J. Kingsman, and S. M. Kingsman, FASEB J. 7:214-222 , 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specifi c but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may med iate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro.