Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro

Nature ◽  
1989 ◽  
Vol 342 (6251) ◽  
pp. 816-819 ◽  
Author(s):  
Thomas J. Daly ◽  
Kathleen Sue Cook ◽  
Gary S. Gray ◽  
Theodore E. Maione ◽  
James R. Rusche
Keyword(s):  
Specific Binding ◽  
Responsive Element ◽  
Hiv 1 ◽  
Rev Protein

Inhibition of Formation of Rev-RRE Complex by Pyronin Y

1993 ◽  
Vol 4 (2) ◽  
pp. 103-111 ◽  
Author(s):  
H. C. Schröder ◽  
H. Ushijima ◽  
A. Bek ◽  
H. Merz ◽  
K. Pfeifer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Complex Formation ◽  
Binding Studies ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Transcript Assembly ◽  
Hiv 1 ◽  
Rev Protein

The interaction of pyronin Y, an RNA intercalating drug, with the binding of Rev protein from human immunodeficiency virus type 1 (HIV-1) to Rev-responsive element (RRE)-containing env RNA was studied. In gel retardation assays, recombinant Rev protein tightly bound to in vitro transcribed RRE RNA. Nitrocellulose-filter-binding studies revealed a dissociation constant of ≈(1–2) = 10−10M (Pfeifer et al., 1991). Pyronin Y efficiently suppressed formation of the Rev-RRE complex. At a concentration of 1 μg ml−1, complex formation was almost completely inhibited. Electron microscopy showed that Rev oligomerizes in the presence of RRE-containing RNA with the formation of short rod-like structures or long filaments, depending on the length of the transcript. Assembly of Rev protein along RRE-containing RNAs was abolished after addition of pyronin Y. Thus pyronin Y represents the first compound described to inhibit Rev-RRE complex formation.

scholarly journals The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

1992 ◽  
Vol 12 (3) ◽  
pp. 1375-1386 ◽  
Author(s):  
D M D'Agostino ◽  
B K Felber ◽  
J E Harrison ◽  
G N Pavlakis
Keyword(s):  
In Vitro Translation ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Utilization Pathway ◽  
Efficient Loading ◽  
Rev Protein

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs

1992 ◽  
Vol 12 (3) ◽  
pp. 1375-1386
Author(s):  
D M D'Agostino ◽  
B K Felber ◽  
J E Harrison ◽  
G N Pavlakis
Keyword(s):  
In Vitro Translation ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Utilization Pathway ◽  
Efficient Loading ◽  
Rev Protein

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.

Characterization of an in vitro-selected RNA ligand to the HIV-1 rev protein

1994 ◽  
Vol 235 (1) ◽  
pp. 237-247 ◽  
Author(s):  
Kirk B. Jensen ◽  
Louis Green ◽  
Sheela MacDougal-Waugh ◽  
Craig Tuerk
Keyword(s):  
Hiv 1 ◽  
Rev Protein

Novel reagents and reactions for drug design

2000 ◽  
Vol 72 (3) ◽  
pp. 347-354 ◽  
Author(s):  
T. J. Baker ◽  
Y. Rew ◽  
M. Goodman
Keyword(s):  
Amino Acid ◽  
Drug Design ◽  
Rna Recognition ◽  
Recognition Element ◽  
Hiv 1 ◽  
Rev Protein

In this presentation, we cover new results from two of our synthetic endeavors: guanidinylation reagents and novel bridged opioids. In the first part, we describe the applications of the diurethane-triflylguanidines to prepare target bioactive structures including peptides, heterocyclic drugs, and aminoglycoside derivatives. The formation of novel guanidinoglycosides led to a family of effective binders to the RNA recognition element of the HIV-1 Rev protein. In the second part, the syntheses of sulfur and amine-bridged cyclic opioids is described. These analogs exhibit enhanced binding, both in vitro and in vivo. Specifically, H-Tyr-c[d-Vall-Gly-Phe-d/l-Alal]-OH (Vall and Alal denote the lanthionine amino acid ends linked by a monosulfide bridge) is potent and highly δ -receptor selective while Tyr-c[(Nγ CH3 )-d-A2 bu-Gly-Phe-NHCH2CH2 -] though nonselective is one of the most potent opioids prepared to date. These molecules are representative of our design of novel peptidomimetic opioids.

scholarly journals The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Damien Morger ◽  
Franziska Zosel ◽  
Martin Bühlmann ◽  
Sara Züger ◽  
Maximilian Mittelviefhaus ◽  
...  
Keyword(s):  
Specific Binding ◽  
Genetic Material ◽  
Correlation Spectroscopy ◽  
Capsid Proteins ◽  
Fold Axis ◽  
Binding Interface ◽  
Species Specific ◽  
Hiv 1 ◽  
B30.2 Domain

ABSTRACTRhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 bothin vivoandin vitro. This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCETRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

scholarly journals 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

1995 ◽  
Vol 308 (2) ◽  
pp. 447-453 ◽  
Author(s):  
S B Renwick ◽  
A D Critchley ◽  
C J Adams ◽  
S M Kelly ◽  
N C Price ◽  
...  
Keyword(s):  
Complex Formation ◽  
Conformational Changes ◽  
Solution Structure ◽  
Cd Spectroscopy ◽  
Sequence Information ◽  
Stem Loop ◽  
Contact Points ◽  
Responsive Element ◽  
Hiv 1 ◽  
Rev Protein

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.

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