The retroviruses HTLV-1 and HTLV-II are distinguished from many other RNA tumor viruses because they encode regulatory proteins that interact with the host cell RNA synthesis machinery. One of these proteins, the tax gene product activates transcription via a target sequence located upstream of the RNA synthesis iniation site in the proviral LTR. The principal objectives of the present work are to elucidate the molecular mechanism underlying tax transactivation and to identify the specific cellular proteins that interact with the tax gene product. Functional Tax protein will be purified from eukaryotic cells infected with a recombinant baculovirus vector. It will be determined whether purified Tax protein binds to or modified any of the previously purified cellular transcription factors that interact with the HTLV-1 LTR, including NF-1, HEF-1T, and various other proteins that bind in or near three 21 bp sequence repeats present in the target region. Binding will be measured directly in coprecipitation and electrophoretic mobility shift assays. In addition, Tax will be incubated with preparations of purified cellular transcription factors to determine whether any of the polypeptides present in these preparations are released from multisubunit complexes or otherwise modified. In separate experiments, purified Tax protein will be added to nuclear extracts from uninfected cells in an attempt to develop a Tax-responsive system for in vitro transcription of the HTLV proviral promoter. As a complementary approach, anti-Tax antibody will be prepared and added to Tax- containing reactions in an attempt to block transcription by steric interference. In addition, a Tax protein affinity column will be prepared and tested for its ability to deplete nuclear extracts of general and promoter-selective transcription factors. Finally, structure-function studies of Tax protein will be initiated. These will include characterization of the differences between Tax protein from HTLV-1 and HTLV-II, using the transcription and protein binding assays described above. Also, these studies will include characterization of a mutant that alters the transactivation specificity of HTLV- I tax to that of HTLV-II tax, two deletion mutants that are transactivation negative, and a mutant that is a transdominant inhibitor of HTLV-II tax. Methods of scaling- up tax expression will also be developed, with the ultimate objective of producing Tax protein in quantities and purity necessary for physical and structural studies.
