Functional significance of the dinucleotide bulge in stem-loop1 and stem-loop2 of HIV-2 TAR RNA

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) encode related proteins called Tat-1 and Tat-2, respectively, that bind directly to the TAR RNA element contained at the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified mechanism. The determinants in the HIV-1 TAR element (TAR-1) that specify binding by the Tat-1 protein have been extensively characterized, while little is known about determinants in the HIV-2 TAR element (TAR-2) that specify binding by the Tat-2 protein. The HIV-2 TAR RNA element (TAR-2) is known to be composed of two stem-loop structures. A dinucleotide bulge is found in each stem of TAR-2 RNA, analogous to the crucial trinucleotide bulge in the single stem-loop of HIV-1 TAR RNA that is the primary binding determinant for binding by the HIV-1 Tat protein. Our results of a nuclease digestion analysis demonstrated that the 5' proximal bulge in TAR-2 is significantly less sensitive to digestion by single-strand specific nucleases than the 3' distal bulge, suggesting that the 5' bulge may be involved in tertiary interaction with other regions of TAR RNA. Deletion of both bulges reduced binding in vitro by the Tat-2 protein and largely abolished transactivation in vivo by Tat-2. Deletion of either bulge alone simplified the pattern of protein/RNA complexes in a gel shift assay, but did not reduce the overall binding affinity of Tat-2. Deletion of the 5' bulge reduced Tat-2 transactivation in vivo to a level approximately 30% that of wild-type TAR-2, while deletion of the 3' bulge had no measurable effect in vivo. Our results suggest that each dinucleotide bulge specifies a Tat-2 binding site, but in the wild-type TAR-2 element the 3' bulge binding site does not appear to be utilized in vivo.