8th International AIDS Conference Amsterdam, Netherlands — July 19-24, 1992

Int Conf AIDS 1992 Jul 19-24; 8:We45 (abstract no. WeA 1004)
Dayton AI, Dayton ET, Konings DA, Powell DM, Bende SM, Shapiro BA, Pantaleo G, Butini L, Maizel JV; NIAID, NIH, Bethesda, MD 20892.

OBJECTIVES: We are pursuing a comprehensive investigation of the rev axis of HIV autoregulation. We report a series of experiments to study the interaction between rev and its viral RNA target sequence, the rev responsive element (RRE), to assess the contribution of cellular factors to the rev axis, and to understand the role of the RRE in contributing to tropism.

METHODS: To study the interaction of the rev protein with the RRE, we asked what are the critical RRE determinants mediating the first, second and third rev binding events. A pool of partially randomized RNA target sequences corresponding to the major rev binding domain of the RRE was bound to rev protein under conditions which give rise to multiple species of complexes corresponding to one, two or three bound rev proteins per RRE. Only the regions near the critical llb domain at the center were randomized. The RNA from these species is being recovered, cloned by PCR and sequenced. To study the cellular factors involved in the rev axis we have formed rev/RRE complexes in the presence of HeLa nuclear extracts. These complexes were analyzed by modified mobility shift assays. To study the effects of the RRE on viral tropism we have constructed a series of proviral clones containing discrete mutations which disrupt localized regions of the RRE without altering the coding potential of the overlapping envelope reading frame. Replication was monitored in a variety of different host cell lines as well as in primary target cells.

RESULTS: We have successfully enriched for pools of rev binding molecules which preferentially bind one, two or three rev proteins. Individual members of these pools are being cloned and sequenced to understand what determines the critical first, second and third rev binding events which precede the subsequent polymerization of rev along the entire RNA molecule. We have reproducibly detected novel complexes formed when rev protein is bound to the RRE in the presence of nuclear extracts. These complexes are not seen when either rev or nuclear extract are omitted from the reaction. Neither are they seen when antisense RRE RNA is used as probe. Data will be presented on the specificity of the reaction and the purification of the activity. We have found that single RRE point mutations can drastically impair replication in some cell types while only slightly delaying replication in others. Some mutations even accelerate viral replication in some cell types while delaying it in others. RNA PCR is being performed to determine the primary mechanism responsible for these effects.

CONCLUSIONS: We will report new determinants critical for the binding of rev to the RRE and will present data on the fractionation and purification of novel cellular factors involved in the rev axis of HIV autoregulation. We will also demonstrate that the RRE can contribute to cell-type specific viral tropism.

Copyright © 1992 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.