The specific hydrolysis of HIV-1 TAR RNA element with the anti-TAR hammerhead ribozyme: structural and functional implications

The main transcriptional regulator of the human immunodeficiency virus is t he Tat protein, which recognises and binds to a fragment RNA at the 5' end of viral mRNA, named transactivation response element (TAR) RNA. Extensive mutagenesis studies have shown that a region of TAR RNA important for Tat b inding involves a set of nucleotides surrounding a characteristic UCU nucle otide bulge. The specific Tat-TAR complex formation enhances the rate of tr anscription elongation but inhibition of that interaction prevents the huma n immunodeficiency virus type 1 (HIV-1) replication. If so, a possibility o f virus inactivation would be a site specific degradation of the TAR RNA el ement. To break down and inactivate TAR RNA, we designated the anti-hammerh ead (HII) ribozyme to cleave nucleosides within the bulge. We showed for th e first time the new type of the AUC hammerhead ribozyme, which hydrolyses specifically the TAR RNA element at C8 nucleotide in the bulge (C24 in the standard TAR RNA numbering). The cleavage reaction has broad magnesium requ irements. Mn and particularly Ca are less efficient. Argininamide interfere s with the cleavage of TAR RNA induced by the ribozyme. These results have two implications; (i) structural, where the HIV-1 TAR RNA element in soluti on occurs in equilibrium of only two forms, one of which, a double stranded RNA, meets structural requirements for ribozyme pairing and cleavage, and (ii) functional, the HH ribozyme can be explored for an inactivation of HIV -1 through the TAR RNA element deintegration. (C) 2001 Elsevier Science B.V . All rights reserved.