Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.

Abstract

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd < 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-anity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long ifiamentous ribonucleoprotein structures containing polymers of rev bound to anking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.Transcription of human immunodeficiency virus type 1 (HIV-1) begins with the production of short fully spliced mRNAs encoding the regulatory proteins tat and rev. As the infection progresses, the combined activity of the rev gene product and a cis-acting RNA sequence called the revresponsive element (RRE) allows the expression of the unspliced virion RNA and partially spliced mRNAs, including the mRNA for the env gene (1-3). It is still unknown whether rev blocks splicing directly (4) or whether it increases the rate of transport of unspliced mRNAs from the nucleus (3).Recent experiments have shown that the interaction between rev and RRE RNA is due to direct binding (5-7). Here we demonstrate that rev binds with high affinity to a purinerich "bubble" containing GG and GUA residues on either side of a double-stranded stem-loop structure present in RRE RNA. Additional rev molecules can then polymerize and form long filamentous ribonucleoprotein structures in association with the RNA sequences that flank the high-affinity binding site. These observations suggest that rev regulates HIV mRNA expression by packaging unspliced mRNA precursors carrying RRE sequences into protein coats, thus preventing access to the splicing machinery. MATERIALS AND METHODSExpression and Purification of Rev. Rev protein was expressed and purified as described (7) or further purified by chromatography on heparin-Sepharose. Rev was applied to heparin columns in a buffer containing 200 mM NaCl/50 mM Tris-HCl, pH 8.0/1 mM dithiothreitol/0.1 mM EDTA/0.1% Triton X-100 and eluted in buffer containing 2 M NaCl. After gel filtration on Superose 6 (prep grade) columns equilibrated with 200 mM NaCl/50 mM Tris HCl, pH 8.0/1 mM dithiothreitol/0.1 mM EDTA, the rev protein appeared homogeneous on SDS/polyacrylamide gels and free of RNA contaminants. Rev concentrations were determined by amino acid analysis of the purified protein.RNA-Binding Assays. DNA inserts containing RRE-related sequences were cloned between the EcoRI (5') and HindIII (3') sites of pGEM-1, either by cloning PCR products or by cloning hybridized pairs of synthetic oligonucleotides. RNA transcripts for binding exper...

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions

“…The unspliced mRNA, which encodes viral structural and enzymatic protein precursors, is e ciently exported from the nucleus only when the viral Rex protein binds to the structured RNA segment entitled Rex-responsive element in the 3'-UTR. Of note, a similar mRNA export system is established in human immunode®-ciency virus type I (HIV-1) by the viral Rev protein and the Rev-responsive element (RRE) (Malim et al, 1989;Zapp and Green, 1989;Heaphy et al, 1991;Malim and Cullen, 1991;Fischer et al, 1994), which plays a key regulatory role in the viral life-cycle (Pomerantz et al, 1992). Interestingly, the Rev-RRE interactions were found to also promote the polysomal assembly and e cient translation of the RRE-containing mRNAs (Arrigo and Chen, 1991;D'Agostino et al, 1992).…”

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region

“…The biological activity of the orthologs of K-Rev observed in exogenous retroviruses, such as H-Rev and HTLV-I Rex, requires the recruitment of these viral regulatory proteins to a discrete primary RNA-binding site that is presented in the context of a larger region of tightly folded RNA termed a "response element" (Hanly et al+, 1989;Malim et al+, 1989Malim et al+, , 1990Zapp & Green, 1989;Bartel et al+, 1991;Heaphy et al+, 1991;Bogerd et al+, 1992)+ Although the primary binding site is clearly essential for biological activity, it is not sufficient and the H-Rev-or Rex-induced export of a target viral RNA therefore requires an almost full-length RNA response element located in cis (Malim et al+, 1989;Huang et al+, 1991)+ Because the biological activity of Rev or Rex response elements is dependent on their folding, these RNA elements can be effectively identified, and their structure predicted, using computer analysis+ Indeed, structures for several retroviral response elements, including both the ;234-nt HIV-1 RRE and the ;254-nt HTLV-I, were first predicted by computer analysis and only subsequently validated by mutational analysis (Hanly et al+, 1989;Malim et al+, 1989;Le et al+, 1990)+ The K-RRE coincides with a predicted unusual RNA folding region Previously, we and others have used functional assays to roughly map the extent of the HERV-K Rev response element (K-RRE) to between residues 8719 and 9152 according to the prototypic HERV-K10 proviral sequence reported by Ono et al+ (1986) (Magin et al+, 1999;Yang et al+, 1999)+ This 434-nt RNA sequence, which is entirely located in the U3 region of the HERV-K LTR, is considerably larger than either the H-RRE or the HTLV-I RxRE, and it therefore appeared likely that the actual extent of the K-RRE would be smaller than this initial estimate+ To gain insight into the true size of the K-RRE, and to determine whether the K-RRE indeed coincides with an unusual folding region (UFR), we used the same computational approach that previously allowed the definition of not only the H-RRE and the HTLV-I RxRE but also the HIV-2 RRE and the Visna-Maedi Virus RRE (V-RRE) (Hanly et al+, 1989;Malim et al+, 1989;Le et al+, 1990;)+ Initially, we focused our attention on sequences extending from 8504 to 9250 within the HERV-K108 proviral clone (Barbulescu et al+, 1999), a sequence that fully includes, but extends significantly beyond, the functionally defined 8719-9152 K-RRE sequence+ Monte Carlo simulations were used to search for any UFR within this HERV-K sequence by calculation of the statistical significance and thermod...…”

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

“…Rev is a viral trans-activator protein that controls the differential expression of viral proteins at the post-transcriptional level by allowing the accumulation in the cytoplasm of unspliced and singly spliced viral RNA containing the Rev-responsive element (RRE) (1)(2)(3)(4)(5)(6)(7). Rev interacts directly with a purine-rich stem-loop within RRE (6,8), and through multimerization additional Rev molecules bind throughout the RRE. In the absence of Rev, the only viral RNA species that accumulate in the cytoplasm are multiply spliced and encode for the viral regulatory proteins.…”