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Probing the details of the HIV-1 Rev-Rev-responsive element interaction: effects of modified nucleotides on protein affinity and conformational changes during complex formation.

ABSTRACT: The solution structure of the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE) has been investigated by enzymic and chemical structural probing of a 71 nt RRE transcript. The minimum sequence information required to maintain recognition by the Rev protein has previously been mapped to a 29 nt stem-loop structure, known as minSLIIB. The key recognition target is a single-stranded RNA bubble at the base of the RNA stem. The fine details of RNA recognition have been probed using chemically synthesized minSLIIBs containing variant base or sugar residues at sites within the bubble. These have been analysed by gel retardation assays and their relative affinities for Rev protein determined. Complex formation between the wild-type minSLIIB RRE and Rev protein was also monitored using CD spectroscopy, which suggests a change in RNA conformation upon Rev binding. The spectral change is consistent with localized melting of RNA, leading to a decrease in the level of base stacking and/or a change in base tilting, during formation of the complex. Deoxynucleotide substitutions on just one side, the 5' side, of the bubble inhibit the conformational change detected by CD. The data are consistent with a dynamic interaction between Rev and its target site. The contact points between Rev and the RRE were probed directly using photo-cross-linking with either ribo-5-bromouridine- or ribo-4-thiouridine-substituted minSLIIBs. The data are consistent with protein-RNA contacts at the bottom of the bubble.

SUBMITTER: Renwick SB

PROVIDER: S-EPMC1136946 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Project description:A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.

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Probing the details of the HIV-1 Rev-Rev-responsive element interaction: effects of modified nucleotides on protein affinity and conformational changes during complex formation.

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