HIV-1 GAG BINDS SPECIFICALLY TO RNA STEM-LOOPS IN THE 5'-LEADER SEQUENCE

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro , with a K-d of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Ga g in an RNA containing nucleotides 325-362. SL4 is predicted by both b iochemical studies and computer folding to be located between nucleoti des 335 and 358. An RNA transcript ending at nucleotide 335 does not b ind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleoti des 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of n ucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At l east two Gag binding sites are therefore located in the leader RNA. Th ose located 5' of nucleotide 335 require the presence of additional 3' sequences. (C) 1998 Elsevier Science B.V. All rights reserved.