Identification of RNA recognition elements for the HTLV-1 Rex protein

Human T-cell leukemia virus type-l (HTLV-I) is the causative agent of Adult T-Cell Leukemia (ATL). Rex is a viral protein essential for HTLV-I replication. It controls the replication of HTLV-I by binding to the Rex Responsive Element (XRE), a large structured RNA. Rex interacts with the XRE to facilitate the appearance of unspliced or singly-spliced mRNAs in the cytoplasm. The unspliced or singly-spliced mRNAs encode viral structural proteins. We have developed RNA molecules that bind to Rex and compete with the XRE. These RNA molecules can potentially inhibit the replication of this virus by preventing the accumulation of structural proteins necessary for viral propagation.; Initial experiments focused on using in vitro selection, a procedure analogous to natural evolution, to identify sequences and structures within the XRE that were required for Rex binding. The essential binding region was found to be similar to the TAR element of the Human Immunodeficiency Virus type-1 (HIV-1) RNA. This sequence similarity suggested that these elements may share common recognition features. A structural model of the XRE binding site is proposed; this model includes a base triple that widens the major groove and allows arginine to interact with an essential guanosine.; Further experiments identified a large family of non-natural sequences and structures that interacted with Rex. The highest affinity structure (anti-Rex aptamer 8-5) was characterized and found to effectively compete with Rex for binding to the XRE. Substituting this sequence for the natural Rex binding site partially restores XRE function in tissue culture cells, suggesting that the unnatural Rex-binding site interacts with Rex in vivo. Additional in vitro selection experiments have identified aptamer sequences and features essential for Rex binding.; We have designed a minimal RNA structure for use in high resolution NMR structural studies. Using in vitro selection and NMR data (in collaboration with Jiang Feng, Sloan Kettering Cancer Center) we have developed a structural model of the anti-Rex aptamer. The model shows a high degree of similarity to BIV-1 TAR RNA.