The regulation of HIV gene expression is dependant on both specific DNA regulatory sequences located in the long terminal repeat which bind cellular transcription factors, and viral regulatory proteins such as tat, rev, and nef. In addition, specific regions of RNA transcribed from different viral regions also appear to be involved in the regulation of viral gene expression. The goal of this grant is to study the stability, processing and transport of the TAR and rev responsive RNA sequences. The HIV TAR region is critical for activation by the tat protein. Using gel retardation and a novel footprinting technique with the nuclease uranyl acetate, we have identified a nuclear protein, SLBP-1, that binds to TAR RNA. Using gel retardation, we have also demonstrated that the tat protein binds to TAR RNA. This suggests that both cellular and viral proteins may bind to HIV RNA. Another region of HIV RNA in the envelope gene known as the rev responsive element has been found to be the site of action for the rev protein. It is possible that either rev alone or in conjunction with cellular proteins binds to RNA transcribed from this region. The goal of this grant is to purify and characterize cellular proteins that bind to both the TAR and rev responsive RNA regions. Furthermore, we will use RNA footprinting to identify regions of these RNAs that bind tat and/or rev. Potential interactions of tat and rev with cellular proteins binding in these two regions will be determined.
The specific aims of this proposal are: (1) to purify and determine the function of the nuclear protein, SLBP-1, that binds to TAR RNA; (2) to determine the site of tat binding to TAR RNA, the domains of tat involved in this binding, and potential interactions of tat with the cellular RNA binding protein; (3) to determine if cellular proteins bind to the rev responsive RNA sequences in the envelope gene and to purify and determine the function of these proteins; (4) to determine if rev binds directly to the rev responsive RNA sequences and/or interacts with cellular RNA binding proteins. These studies will be important in determining the role of both cellular and viral proteins in the regulation of HIV RNA processing, stability, transport, and translation. Such regulation is likely one determinant in maintaining HIV latency.
|Harrich, D; Garcia, J; Mitsuyasu, R et al. (1990) TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes. EMBO J 9:4417-23|