The rev protein of HIV is a 19 kD protein, that has been shown to specifically regulate the production of structural proteins in infected cells. Rev is absolutely essential for virus replication and functions to facilitate nuclear export of the gag/pol and env mRNAs. Without rev these mRNAs accumulate in the nucleus. The rev protein has been shown to act through a highly structured rev responsive element (RRE), that is present in the env and gag/pol mRNAs of both HIV1 and HIV2. However, the detailed mechanism behind the rev function is still totally unknown. Surprisingly the HTLV rex proteins can functionally substitute for rev to obtain HIV structural protein expression. It is the goal of the experiments proposed in this program to gain further understanding of the mechanisms underlying the function of the rev protein. We will also collaborate closely with the drug screening core to adapt our assay systems for rev function for drug screening purposes.
The specific aims are: 1) To produce and purify large amounts of functional HIV rev proteins and HTLV rex proteins. These proteins will be used in binding experiments and functional studies. In a collaboration with Dr. M. Rossmann, Purdue University, attempts will be made to crystallize the HIV1 rev protein. 2) To study the interactions that take place between rev, rex and cellular components and the RRE in vitro and in vivo. For this we will use purified proteins and cellular extracts. 3) To further analyze rev and rex regulation of HIV expression in functional assays and to map the specific regions of the HIV1 and 2 RREs that are essential for rev and rex function. 4) To analyze whether the RNA that is a substrate for rev and rex, is complexed to U1 snRNA and/or other components of the normal cellular splicing machinery. 5) To evaluate the effect of RNA and deoxyoligonucleotides that are antisense with respect to the RRE, on rev and rex function.
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