Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1

Abstract

We have analyzed the action of the Rev and Tev proteins of human immunodeficiency virus type 1 (HIV-1) and of the Rex protein of human T-cell leukemia virus type I (HTLV-I) on a series of Rev-responsive element (RRE) mutants. The minimum continuous RRE region necessary and sufficient for Rev function was determined to be 204 nucleotides. Interestingly, this region was not sufficient for Tev or Rex function. These proteins require additional sequences, which may stabilize the structure of the RRE or may contain additional sequence-specific elements. Internal RRE deletions revealed that the targets for Rev and Rex can be separated, since mutants responding to Rev and not Rex and vice versa were identified. Tev was active on both types of mutants, suggesting that it has a more relaxed specificity than do both Rev and Rex proteins. Although Rev and Rex targets within the RRE appear to be distinct, the trans-dominant mutant RevBL prevents the RRE interaction with Rex. RevBL cannot inhibit the function of Rex on RRE deletions that lack the Rev-responsive portion. These results indicate the presence of distinct sites within the RRE for interaction with these proteins. The binding sites for the different proteins do not function independently and may interfere with one another. Mutations affecting the RRE may change the accessibility and binding characteristics of the different binding sites.

Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex

“…2). In agreement with recent studies (1,39), the Rev protein interacted with RRE stem-loop II while stem-loop IV/V was the target site for the Rex protein (Fig. 2, lanes 10 and 11 versus lanes 18 and 19).…”

“…2, lane 12). This result indicates that HTLV-I Rex amino acid residues located outside of the NLS functionally contribute to the binding of Rex to RRE stemloop IV/V since the Rev RNA binding domain is by itself unable to recognize this target RNA sequence (1,39). Thus, the functionally equivalent HIV-1 Rev and HTLV-I Rex trans-activators display a different domain organization with respect to subcellular localization and RNA recognition.…”

“…Additional studies revealed that the 189-aminoacid wild-type Rex protein interacts with an RRE substruc-* Corresponding author. ture (stem-loop IV/V) which is different from the one recognized by the homologous Rev protein (stem-loop II) (1,39). These different cis-acting RNA sequences enabled us to functionally characterize our mutant chimeric Rev-Rex proteins.…”

“…1B, lanes 2 and 3) (7,24). Clearly, specific signals were also detectable for the Rev-Rex hybrid proteins ( [33][34][35][36][37][38][39][40][41][42][43][44][45][46] Rex[vNLS [33][34][35][36][37][38][39][40][41][42][43][44] Rex[vNLS [35][36][37][38][39][40][41][42][43][44][45][46] were obtained with Rex[vNLS33-44] and the Rex protein encoded by RexANLS (Fig. 1C, panels D and G).…”

The human T-cell lymphotropic virus type 1 Tof protein contains a bipartite nuclear localization signal that is able to functionally replace the amino-terminal domain of Rex

“…The p13 II protein was expressed from plasmid pLsp13AU, which contained nt 6919 to 7193 of CS-HTLV-I, followed by a sequence coding for the AU-1 epitope. The Rex protein was expressed from pL3Rex (58) or from the natural tax/rex mRNA, using plasmid BSTR-3, which contained the viral 5Ј LTR and exons 1, 2, and 3 (including the 3Ј LTR) of CS-HTLV-I, cloned into pBluescript. The Tof coding sequence was altered by site-directed mutagenesis (30) using as a template pTxTof, a pBluescript-derived plasmid containing a DNA fragment (nt 4820 to 4831 linked to nt 6478 to 7223) spanning the Tof ORF; resulting mutated sequences were transferred to LdKL3pA, generating the plasmids specified in the legend to Fig.…”