A MOLECULAR RHEOSTAT - COOPERATIVE REV BINDING TO STEM-I OF THE REV-RESPONSE ELEMENT MODULATES HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 LATE GENE-EXPRESSION

The complete biologically active human immunodeficiency virus type-1 ( HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in lengt h and includes an extra 58 nt on the 5' end and 59 nt on the 3' end be yond the sites proposed m the original models for the RRE secondary st ructure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protec tion experiments have shown that rev binds to restricted regions of th e RRE, including the high affinity site located at the lease of Stem I Ib and along the length of the Stem I region. The three large stem-loo p structures which protrude from Stem I and Stem IIb (Stems IIc, III IV and V) remain accessible to nucleases even in the presence of a la rge excess of protein. Gel-retardation experiments show that the trunc ations of Stem I reduced the total number of rev molecules that can bi nd co-operatively and with high affinity to the RRE RNA. To test wheth er the elongated Stem I structure is required for maximal rev activity , a series of truncations which progressively reduced the length of St em I was introduced into an HIV-1 derived reporter plasmid. In the pre sence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat a nd rev mRNBs. Each of the truncations in Stem I reduced the rev respon ses, with the longest truncations producing the greatest losses of act ivity. The data suggest that the RRE acts as a ''molecular rheostat'' designed to detect rev levels during the early stages of the HIV growt h cycle.