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 re lated proteins called Tat-1 and Tat-2, respectively, that bind directl y to the TAR RNA element contained at the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified me chanism. The determinants in the HIV-1 TAR element (TAR-1) that specif y binding by the Tat-1 protein have been extensively characterized, wh ile 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 dinucl eotide bulge is found in each stem of TAR-2 RNA, analogous to the cruc ial 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 prote in. Our results of a nuclease digestion analysis demonstrated that the 5' proximal bulge in TAR-2 is significantly less sensitive to digesti on by single-strand specific nucleases than the 3' distal bulge, sugge sting that the 5' bulge may be involved in tertiary interaction with o ther regions of TAR RNA, Deletion of both bulges reduced binding in vi tro by the Tat-2 protein and largely abolished transactivation in vivo by Tat-2, Deletion of either bulge alone simplified the pattern of pr otein/RNA complexes in a gel shift assay, but did not reduce the overa ll binding affinity of Tat-2. Deletion of the 5' bulge reduced Tat2 tr ansactivation in vivo to a level approximately 30% that of wild-type T AR-2, while deletion of the 3' bulge had no measurable effect in vivo. Our results suggest that each dinucleotide bulge specifies a Tat-2 bi nding site, but in the wild-type TAR-2 element the 3' bulge binding si te does not appear to be utilized in vivo. (C) 1994 Academic Press, In c.