INTERACTION OF HIV-1 TAT PROTEIN WITH HEPARIN - ROLE OF THE BACKBONE STRUCTURE, SULFATION, AND SIZE

Human immunodeficiency virus type 1 (HIV-1) Tat protein is released fr om infected cells. Extracellular Tat enters the cell where it stimulat es the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Ben elli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialu nga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) an d transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10; 1733-1739) activity of extracellular Tat. Here we demonstrate that hep arin binds specifically to recombinant HIV-1 Tat produced as glutathio ne S-transferase (GST) fusion protein add immobilized on glutathione-a garose beads. Heparin and heparan sulfate (HS), but not dermatan sulfa te, chondroitin sulfates A and C, hyaluronic acid, and K5 polysacchari de, competed with H-3-labeled heparin for binding to immobilized GST-T at and inhibited HIV-LTR transactivation induced by extracellular GST- Tat. Selective 2-0-, 6 O-, total-O-desulfation, or N-desulfation/N-ace tylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduce d capacity to inhibit the transactivating activity of GST-Tat. Very lo w molecular weight heparins showed a significant decrease in their cap acity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kD a heparin was affinity chromatographed on immobilized GST-Tat to isola te binding and non-binding subfrac tions, the Tat-bound fraction was g reater than or equal to 1,000 times more potent than the unbound fract ion in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with hepari n/HS and that high affinity Tat heparin interaction requires at least some 2-O, 6-O-, and N-positions to be sulfated. The Tat binding activi ty of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating t he possibility to design specific heparin/HS-like structures with Tat- antagonist activity.