Human T-cell leukemia viruses types I (HTLV-I) and II (HTLV-II) and human immunodeficiency viruses types I (HIV-1) and II (HIV-2), are distinct but related human retroviruses that have been associated with human disease. These retroviruses have evolved similar trans-acting regulatory proteins, HTLV-I and -II Rex and HIV-1 and -2 Rev, required for productive viral infection. Rex and Rev regulate viral RNA processing and the switch from viral regulatory to viral structural gene expression. In addition, these viruses have evolved cis-acting RNA response elements which fulfill analogous roles in mediating gene regulation by Rex and Rev. We have previously identified a cis-acting Rex responsive element (RxRE) and adjacent inhibitory sequences, or cis-acting repressive sequences (CRS), in HTLV-II 5' long terminal repeat (LTR) RNA and have demonstrated both that Rex protein binds to RxRE RNA and that binding correlates with Rex function. We wish to define further the mechanisms of action of HTLV-II Rex, by focusing on the role of cis-acting viral RNA response elements and of cellular factors in Rex regulation. In addition, we wish to determine whether these cellular factors also have effects on Rev regulation in HIV-1.
The Specific aims are: 1.) To identify all CRS and RxRE elements within HTLV-II RNA by testing in a transient transfection gene expression assay, both heterologous vectors and proviral constructs containing deletions or nucleotide substitutions in HTLV-II transcribed sequences. 2.) To determine the mechanisms by which CRS and RxRE elements affect viral gene expression by analyzing effects on viral RNA processing, with or without Rex in trans, at several stages: RNA stability, RNA splicing, RNA transport from nucleus to cytoplasm, and RNA polysome association. 3.) To identify and clone cellular proteins that are involved in the regulation of viral gene expression by Rex and to determine how Rex interacts with those cellular proteins using in vitro binding assays and lambdagt11 expression library screening or a biotinylated RNA-avidin purification procedure. 4.) To characterize the crossover regulatory effects on HIV-1 gene expression of cellular proteins that affect HTLV-II Rex regulation of HTLV-II gene expression. This proposal addresses the role of cellular factors in Rex and Rev regulation of viral gene expression. Characterization of these factors may lead to a better understanding of the regulation of cellular RNA processing, and may reveal novel targets for antiretroviral therapy.
