A Solution to Limited Genomic Capacity: Using Adaptable Binding Surfaces to Assemble the Functional HIV Rev Oligomer on RNA

Daugherty, Matthew D.; D’Orso, Iván; Frankel, Alan D.

doi:10.1016/j.molcel.2008.07.016

Cited by 109 publications

(268 citation statements)

References 46 publications

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“…Next, more Rev molecules (about 6-12) bind co-operatively along the other regions of the RRE in a process that involves protein-protein as well as protein-RNA interactions (41,46,51,52,62,104,149,155,249,250). Rev binding to the primary Rev binding site and Rev oligomerization on the RRE are both essential for Rev-RRE function (47,149,168,249).…”

Section: Nuclear Export Of Viral Mrnas and The Rev-rre Pathwaymentioning

confidence: 99%

“…Rev binding to the primary Rev binding site and Rev oligomerization on the RRE are both essential for Rev-RRE function (47,149,168,249). While the former allows the Rev to make specific contacts with the RRE (13), the latter increases the affinity of Rev-RRE complex by ~500 fold (51). Therefore, it appears that the tight cooperative nature of Rev-RRE binding explains how Rev manages to specifically and preferentially bind to the RRE RNA in the presence of many other RNAs within the cell.…”

Section: Nuclear Export Of Viral Mrnas and The Rev-rre Pathwaymentioning

confidence: 99%

“…A series of genetic, biochemical, and biophysical studies have contributed to our understanding of Rev structure and some molecular details of the assembly of NL4-3 Rev and the RRE (12,13,49,50,53,116,118,154,185,228). For a long time, the only structural information of Rev came from an NMR study (13) and circular dichroism experiments (225) using a 23 amino acid synthetic polypeptide corresponding to the ARM domain of Rev (residue 34 to 50) bound to the stem IIB of RRE containing the high affinity Rev binding site (13).…”

Section: Rev Structure and Functional Domainsmentioning

confidence: 99%

“…The authors (51) found that, to make contact with this RRE site, Rev uses an ARM interface (Arg38, Arg41, and Arg46) that is different from that used to make contact with the high affinity stem IIB region. When each of these residues was mutated, the interaction between the secondary Rev binding site and ARM peptide was disrupted but the same mutations did not affect stem IIB binding.…”

Section: Rev Structure and Functional Domainsmentioning

confidence: 99%

“…Functionally, the RRE has been modeled as a scaffold for binding Rev molecules (48). The primary Rev binding site on the RRE has been mapped to stem IIB (41,56,125), and stem I/I' has been shown to provide secondary Rev binding sites (51,155).…”

Section: Rationalementioning

confidence: 99%

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Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

Sherpa¹

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HIV-1 is a retrovirus of global health interest as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). It is also used as a molecular tool to study various eukaryotic cellular processes. For instance, major discoveries on the Crm1 cellular nuclear export were made using the HIV Rev protein and its RNA binding partner, the Rev Response Element (RRE). The RRE is a cis-acting RNA element with multiple stem-loops present in all intron-retaining HIV mRNAs. Binding of the Rev protein to the primary binding site, and Rev multimerization on other regions of the RRE, is required for the nucleo-cytoplasmic export of these mRNAs. This is an essential step in HIV replication. However, the precise secondary structure of the HIV-1 RRE remains controversial. Studies have reported that the RRE has either 4 or 5 stem-loops, which differ only in the rearrangement of regions that lie outside of the primary Rev binding site. To understand the role played by these regions, we have examined the relationship between these two structures and Rev-RRE activity.In vitro transcribed NL4-3 RRE was found to migrate as a "doublet" band on a native polyacrylamide gel. Using in-gel Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE), we found that one of these bands consisted of an RRE with a 5 stem-loop structure, whereas the other was a 4 stem-loop structure. Thus, our data demonstrate, for the first time, that the NL4-3 RRE exists in two alternative structures.To study the significance of these alternative structures, we made RRE mutants predicted to allow only one or the other of the structures to form. The predictions were confirmed using SHAPE. We then compared the activity of the two forms to each other and to the wt RRE. Analysis of the complexes that each RRE formed with purified Rev protein in vitro showed no significant difference in Rev binding affinity between the two structures. However, we observed differences in the migration rates of these complexes on native gels, suggesting structural differences. The RREs were also tested for their abilities to promote viral replication, by inserting each RRE into the Nef region of an RRE-defective provirus which contained RRE mutations that do not change the Env protein.Growth kinetics and competition assays showed that the virus with the 5 stemloop RRE had a higher replicative fitness than the virus with either the wt or the 4 stem-loop RRE. Between the wt and the 4 stem-loop RRE-containing viruses, the virus with the wt RRE appeared more fit. These results suggest that HIV may use two alternative RRE secondary structures to modulate replication, potentially allowing adaptation to environmental demands in time and/or space, analogous to the use of riboswitches in bacteria.

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Section: Nuclear Export Of Viral Mrnas and The Rev-rre Pathwaymentioning

confidence: 99%

Section: Nuclear Export Of Viral Mrnas and The Rev-rre Pathwaymentioning

confidence: 99%

Section: Rev Structure and Functional Domainsmentioning

confidence: 99%

Section: Rev Structure and Functional Domainsmentioning

confidence: 99%

Section: Rationalementioning

confidence: 99%

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Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

Sherpa¹

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Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

et al. 2013

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Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

et al. 2013

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Numerous antibiotics bind to ribosomal RNA, and many functional RNA motifs have considerable therapeutic potential. However, the development of small RNA-binding agents has been hampered by the difficulties posed by these structures, which have limited physicochemical diversity and are often flexible.[1] In order for this approach to be successful, it is essential to identify new chemical scaffolds recognizing RNA.The Rev response element (RRE) is a strongly conserved 350-nucleotide structure located in the env gene of human immunodeficiency virus type-1 (HIV-1) RNA. Within subdomain IIB of the RRE, the unusually widened major groove of a 4:6 internal loop forms a high-affinity complex [2] with the arginine-rich a-helix of Rev, a virally encoded 116 amino acid protein that adopts a helix-turn-helix conformation [3] ( Figure 1). This interaction between internal loop IIB and the RNA-binding a-helix of Rev (Rev 34-50 ) is essential for virus viability because it triggers a cascade of events that allow the transport of unspliced or incompletely spliced viral RNA molecules into the cytoplasm of the infected cell in the late phase of the viral infectious cycle. These events include the cooperative incorporation of additional Rev molecules into the complex through interactions with further sites on the RRE and protein-protein contacts, [4] and the tethering of the RRE-Rev ribonucleoprotein to the Crm1 host export factor. In addition to RNA nuclear export, Rev has been shown to enhance translation and packaging [5] and to control the nucleocytoplasmic shuttling of the HIV-1 integrase.[6] Clearly, this protein represents a pivotal target for HIV-1 therapy, but to date, the development of Rev-based inhibitors has remained an elusive goal. Herein we report the structurebased design of new RNA-binding p-terphenyl Rev mimics that inhibit RRE-Rev function and HIV-1 replication.We aimed to generate organic ligands that mimic the three-dimensional distribution of the side chains of Rev 34-50 complexed with internal loop IIB of the RRE [2] (Figure 1 a). Some reports had shown that tris-3,2',2''-substituted p-ter- Figure 1. Structure-based design of RNA-binding p-terphenyls. a) View of the complex formed by internal loop IIB of the RRE and Rev 34-50 . The a-helix (red) is deeply embedded in its RNA receptor (gray). b) Representation of a bilaterally substituted p-terphenyl molecule (green) superposed on an a-helix (red). c) Chemical structure and synthesis strategy of a 3,5,2',6',2'',6''-substituted p-terphenyl molecule. d) Secondary structures of the RNA oligonucleotides utilized in this study:

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A Solution to Limited Genomic Capacity: Using Adaptable Binding Surfaces to Assemble the Functional HIV Rev Oligomer on RNA

Cited by 109 publications

References 46 publications

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

Structure‐Based Design of an RNA‐Binding p‐Terphenylene Scaffold that Inhibits HIV‐1 Rev Protein Function

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