Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element.

Abstract

The specific interaction between a defined structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.The human retroviruses have proven to be an excellent system to study the complex regulation of gene expression in mammalian cells. Human immunodeficiency virus type 1 (HIV-1), which is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) (1, 2), has at least two novel genetic regulatory mechanisms. These are mediated by the virus-encoded regulatory proteins Rev and Tat, which are essential for viral replication (3). Whereas Tat appears to act on transcriptional initiation and elongation (3), Rev functions posttranscriptionally. Rev is necessary for the production of viral structural proteins (4, 5) through regulation of the cytoplasmic appearance of unspliced or singly spliced viral mRNA (6-8). Rev functions either directly or indirectly at the level of splicing (5, 6, 9, 10). Rev action requires the presence of a cis-acting RNA target sequence termed the Rev response element (RRE), which is located in the envelope gene of HIV-1 (8, 11-13). The minimal sequence required for full Rev response in vivo has been mapped to a 234-nucleotide region that has the potential of forming four stem-loops (I, III, IV, and V) and one branched stem-loop (IIA, IIB, and IIC) secondary structure (8). Moreover, stem-loops III, IV, and V can be deleted without abolishing the Rev response in vivo (11,14,15 MATERIALS AND METHODS Construction of Recombinant Baculovirus Expressing HIVRev. The Stu I-HindIII fragment of the plasmid pcREV (24) containing the Rev coding region was cloned into the BamHI site of the baculovirus expression vector pVL941 (25) by standard techniques. The resulting plasmid, pVREV, was cotransfected with wild-type baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) DNA into Sf9 insect cells and a recombinant baculovirus expressing Rev was plaque-purified (26). Purification of Recombinant HIV Rev from Infected InsectCells. Large-scale infections of insect cells were performed as described (27). Infected cells were harvested 48-72 hr postinfection and washed twice with phosphate-buffered saline. The cell ...

Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element

“…Furthermore, the structure of the RmRE may be influenced by its flanking sequences, by co-transcriptional folding, or by other features of the cellular environment that are not captured in vitro in structure mapping experiments. Nevertheless, the central features of the predicted structure, the extensive secondary structure, multiple stem-loops, and long-range base pairs, are supported by boundary mapping experiments and are consistent with results from other complex retroviruses (15,16,18,26), suggesting that these features are likely to be present in the natural context of the RmRE.…”

“…The published RmRE structure, which was solely based on the Mfold algorithm (11,28), contains predominant double-stranded regions typical of constitutive transport elements found in simple retroviruses (34 -37). In contrast, our constrained structure has many single-stranded regions and a complex stem-loop structure that more closely resembles the response elements of the complex retroviruses, HIV, human T-cell leukemia virus, and human endogenous retrovirus type K (13,16,26,30,38). Recent work has shown that the HIV Rev and human T-cell leukemia virus Rex regulatory proteins can function on the MMTV reporter vector pHMRluc in human cells (24).…”

“…RNase T1 cleaves preferentially at singlestranded G residues, RNase A cleaves at single-stranded C or U residues, and RNase V1 primarily digests base-paired nucleotides without strong preferences for nucleotide identity (25). Thus, RNase probes used in combination give extensive information on base pairing status throughout a structured RNA (26,27).…”

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

“…Although several in vitro studies have suggested that Rev multimerization is facilitated by interaction with RRE (16,39,(61)(62)(63), it has remained unclear whether multimerization occurs prior to or subsequent to RRE binding. Comparison of the dissociation constant of Rev-GFP and RevM5-GFP in the nucleolus when bound to Rev-BFP revealed that the interaction of Rev-GFP with the nucleolus is stronger than its multimerization.…”

Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element

“…Analysis of the native RRE confirms four structured regions: I, IIA/IIB/IIC, and III/IV and V (19,20). Region I comprises a single large anchoring stem spanning nts 60-95 and 253-291.…”