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.

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.

Modeling and solution structure probing of the HIV-1 TAR stem-loop

1993 ◽  
Vol 11 (2) ◽  
pp. 92-97 ◽  
Author(s):  
Andrew D. Critchley ◽  
I. Haneef ◽  
Diane J. Cousens ◽  
Peter G. Stockley
Keyword(s):  
Solution Structure ◽  
Stem Loop ◽  
Structure Probing ◽  
Hiv 1

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region

Cell ◽  
1990 ◽  
Vol 60 (4) ◽  
pp. 685-693 ◽  
Author(s):  
Shaun Heaphy ◽  
Colin Dingwall ◽  
Ingemar Ernberg ◽  
Michaet J. Gait ◽  
Sheila M. Green ◽  
...  
Keyword(s):  
Loop Structure ◽  
Response Element ◽  
Stem Loop ◽  
Rev Response Element ◽  
Stem Loop Structure ◽  
Hiv 1 ◽  
Rev Protein

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

NMR solution structure of the RNA-binding peptide from HIV-1 Rev protein

Biochemistry ◽  
1995 ◽  
Vol 34 (26) ◽  
pp. 8242-8249 ◽  
Author(s):  
Martin J. Scanlon ◽  
David P. Fairlie ◽  
David J. Craik ◽  
Darren R. Englebretsen ◽  
Michael L. West
Keyword(s):  
Solution Structure ◽  
Rna Binding ◽  
Nmr Solution Structure ◽  
Binding Peptide ◽  
Hiv 1 ◽  
Rev Protein

Giant Human T-Lymphocytes (Jurkat Cells) Expressing HIV-1 Tat-72: LM and TEM.

1997 ◽  
Vol 3 (S2) ◽  
pp. 15-16
Author(s):  
J. Gilloteaux ◽  
J. Koch ◽  
J. M. Jamison ◽  
M. Authelet ◽  
C. Sowick ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Transcriptional Activation ◽  
Jurkat Cells ◽  
Stable Cell Line ◽  
Stem Loop ◽  
Human T Lymphocytes ◽  
Stem Loop Structure ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Hiv 1

The human immunodeficiency virus (HIV-1) is dependent upon the synthesis of TAT for efficient transcriptional activation and replication. Following translocation to the nucleus TAT interacts with a cis-acting TAT-responsive element located between nucleotides +1 and +42 within the HIV-1 long terminal repeat. This region contains an RNA stem loop structure TAR which interacts with TAT and cellular proteins to increase transcription 1000 fold We have transfected human T-lymphocytes with an expression vector containing the HIV-1 LTR driving the synthesis of TAT-72. A stable cell line was generated by selection with G418. Cells were grown on round glass coverslips coated with a solution of fibronectin overnight (at 37 °C). In addition to light microscopy (LM) observations, cells on coverslips were fixed with a 3.5% glutaraldehyde buffered solution (0.1 M Na cacodylate) for 5 min at room temperature and gently centrifliged in a microfuge (400 rpm, 3min) to obtain a cellular pellet.

Human T-lymphocytes (Jurkat cells) expressing HIV-1 TAT-72: SEM

1995 ◽  
Vol 53 ◽  
pp. 1014-1015
Author(s):  
J. Koch ◽  
J. Gilloteaux ◽  
J. M. Jamison ◽  
J.L. Summers
Keyword(s):  
T Lymphocytes ◽  
Transcriptional Activation ◽  
Sucrose Solution ◽  
Jurkat Cells ◽  
Stable Cell Line ◽  
Stem Loop ◽  
Cis Acting ◽  
Human T Lymphocytes ◽  
Responsive Element ◽  
Hiv 1

The human immunodeficiency virus (HIV-1) is dependent upon the synthesis of TAT for efficient transcriptional activation and replication. Following translocation to the nucleus TAT interacts with a cis-acting TAT-responsive element located between nucleotides +1 to +42 within the HIV- 1 long terminal repeat. This region contains an RNA stem loop structure TAR, which interacts with TAT and cellular proteins to increase transcription 1000 fold.We have transfected human T-lymphocytes with an expression vector containing the HIV-1 LTR driving the synthesis of TAT-72. A stable cell line was generated by selection with G418. Cells were grown on round glass coverslips coated with a solution of fibronectin overnight (at 37°C). In addition, cells on coverslips were fixed with a 3.2% glutaraldehyde buffered solution (0.1M Na cacodylate) for 5 min at room temperature, then 15 min at 4°C, then washed gently in a buffer-sucrose solution. Postfixation was performed with an aqueous mixture containing 1.5% OsO4+ 0.5% RuO4 for 20 min at 18°C.

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