Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA.

Abstract

A region of potential complex secondary structure within the human immunodeficiency virus env mRNA has been implicated in Rev-mediated export of viral structural mRNAs from the nucleus to the cytoplasm. By using an RNase protection gel-mobility-shift assay, we demonstrate that purified Rev protein forms a stable complex with this Rev-responsive RNA. RNAs with mutations designed to disrupt formation of a predicted stem structure no longer interact with Rev. However, Rev binding is restored upon annealing of the two complementary RNAs that make up the stem. These results suggest that direct interaction of Rev with the Rev-responsive element could facilitate transport of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm. The rev gene, which is conserved in HIV-2 (8) and simian immunodeficiency virus (8, 9), encodes a small phosphorylated (10,11) nuclear protein (12) that accumulates in the nucleolus (13,14). Synthesis ofRev is required for expression of the viral structural genes (i.e., gag, pol, and env), whereas expression of the nonstructural genes (i.e., tat, vpu, and nef) proceeds in its absence (3,4,15). Studies indicate that the block in structural gene expression, in the absence of Rev, results from entrapment of mRNA in the nucleus (16). Although the mechanism that governs this nuclear mRNA sequestration and subsequent Rev-mediated export remains to be determined, results of several independent studies, using different assays to measure Rev function, indicate the presence of "negative" sequences that presumably govern retention of viral mRNA in the nucleus (17-21) and a distinct Rev-responsive element (RRE) (16,17,19,21) that is required for the Rev-mediated export of nuclear mRNA to the cytoplasm. RRE was originally mapped to nucleotides (nt) 7202-7720 (17) and was referred to as CAR for cis-acting antirepression sequence (18). More recent studies have further localized this RRE (16) to near nt 7300-7539, a region that is predicted to have a high degree of secondary structure.In dithiothreitol/0.5 mM phenylmethylsulfonyl fluoride, stirred overnight, and then disrupted using a Manton Gaulan apparatus. Cellular debris was removed by a brief centrifugation (10,000 x g for 15 min) and the supernatant was adjusted to 0.5% polyethylenimine. Precipitated nucleic acids were pelleted by centrifugation and the coprecipitated Rev protein was released by resuspension of the pellet in 3 M KCl. The resuspended material was then adjusted to 20% (wt/vol) ammonium sulfate and the precipitate, which was found to have the greatest proportion ofRev, was dissolved in isotonic phosphate-buffered saline containing 0.5 M KCl. The mixture was then applied directly to a Sepharose 4B anti-Rev monoclonal antibody immunoaffinity column. The column was prepared using anti-Rev monoclonal antibody at 5 mg/ml of affinity matrix as described by the manufacturer (Pharmacia). Elution and binding conditions were also as suggested by the manufacturer. The anti-Rev monoclonal antibodies were prepared again...

Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids.

“…We and others have shown recently that purified Rev protein derived from E. coli forms a specific interaction with RRE RNA {Daly et al 1989;Zapp and Green 1989;Cochrane et al 1990a;Heaphy et al 1990;Malim et al 1990). Because of possible difficulties associated with purification of each of the 14 mutations described, the feasibility of using Rev within E. coli lysates in the binding assays was examined.…”

“…Western blot analysis with an anti-Rev antiserum raised against a synthetic peptide corresponding to amino acids 1-20 (residues not affected by the mutagenesis) showed comparable levels of Rev protein in each lysate (data not shown). One microliter of cleared lysate was incubated with in vitro-transcribed RRE RNA transcripts, and binding was measured in a qualitative gel mobility-shift ribonuclease T1 protection assay (Cochrane et al 1990a). As shown in Figure 3, many of the Rev mutants failed to interact with the RRE RNA.…”

“…Expression of Rev is required for the export of HIV structural mRNA from the nucleus to the cytoplasm (Emerman et al 1989;Felber et al 1989;Hammarskj61d et al 1989;Malim et al 1989b). Recent studies demonstrate that the Rev proteins encoded by both HIV-1 and HIV-2 interact with a segment of RNA present within their respective env gene messages (Daly et al 1989;Zapp and Green 1989;Cochrane et al 1990a; P.J. Dillon, P. Nelbock, A. Perkins, and C.A.…”

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.

“…In addition, bulged adenosines are present in the 16S rRNA binding sites of ribosomal proteins $8 and $15 (Garrett et al 1984). The trans-acting factor Rev of HIV-1, which is essential for the production of unspliced (-~9 kb) and singly spliced (~-4 kb)transcripts that encode viral structural proteins (for review, see Cullen andGreene 1989 andFelber 1990), was recently reported to be an RNA-binding protein (Daly et al 1989;Zapp and Green 1989;Cochrane et al 1990;Heaphy et al 1990). Its target, the Rev responsive element (RRE), forms a complex secondary structure (Dayton et al 1989), that was shown to be necessary for both Rev binding in vitro and the Rev response in vivo (Malim et al 1990;Olsen et al 1990).…”

The Human T-cell leukemia virus Rex protein

“…However, mutating certain arginine residues in the NLS alters the nucleolar but not the nuclear localization of Rex. Several studies indicate that targeting of Rex to its correct subcellular compartment is critical for its proper function (69,70).…”