HIV-1 gene expression is dependent on distinct regulatory elements in the long terminal repeat that serve as binding sites for cellular transcription factors. The viral transactivator protein tat is able to activate gene expression from the HIV-1 LTR by stimulating transcriptional elongation. A regulatory element between +1 and +60 in the HIV-1 LTR which is capable of forming a stable stem-loop RNA structure designated TAR is critical for tat activation. Three elements within TAR RNA including the upper stem structure, a three nucleotide bulge, and a six base pair loop are all important for tat activation. Tat protein directly binds to the TAR RNA bulge sequences, and recent data suggests that cellular factors binding to TAR RNA regions such as the loop may be important for tat function. The goal of this grant is to characterize the function of cellular factors that bind to the HIV-1 TAR RNA loop sequences. The model that we wish to test is that tat in conjunction with cellular factors binding to TAR RNA are able to activate HIV-1 gene expression by either direct or indirect interactions with transcription initiation or elongation factors. We have purified a cellular factor that we designated TRP-185 and have recently cloned the gene encoding this factor. TRP-185 binds directly to the HIV-1 TAR RNA loop sequences though its binding is dependent on a class of cellular proteins designated TRP-185 associated cofactors. Analysis of the TRP-185 gene indicates that it encodes a novel protein which has interesting homology with procaryotic sigma factors. We have also purified these cofactors and cloned the genes of three cofactor proteins designated CF-1, CF-2, and CF-3. The CF-1 gene contains a motif similar to those found in a variety of RNA binding proteins, CF-2 has homology with a group of proteins which have GTPase activity, and CF-3 has homology with potential ATPases or protein kinases. Now that we have cDNA clones encoding these genes, we will study their role in mediating basal and tat-induced gene expression of the HIV-1 LTR.
The specific aims of this proposal are: (1) To overexpress and purify wild- type and mutated forms of the TRP-185 protein using vaccinia expression vectors in an attempt to define domains of TRP-185 that are involved in TAR RNA binding and transcriptional activation; (2) To determine the mechanism by which cofactor proteins are able to facilitate TRP-185 binding to TAR RNA and to determine if tat modifies this process; (3) To identify cellular proteins that directly interact with TRP-185 and determine if these proteins influence either its binding properties or transcriptional activation; and (4) To use in vitro transcription assays with purified RNA polymerase II and general transcription factors to identify potential cellular targets for TRP-185 and tat. These studies will be important in understanding the function of cellular factors that bind to TAR RNA and determining their role in modulating tat function.
| 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 |
