Abstract
Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a Ka = 16.2±0.6×107 M−1 where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (Ka = 3.1±0.4×107 M−1) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.
Highlights
The large pre mRNA transcript produced by HIV-1, during its early phase of lifecycle, undergoes alternative splicing to generate small mRNA transcripts for production of certain regulatory proteins
In order to detect structural changes upon binding of Rev and RSG-1.2 peptide, Circular dichroism (CD) spectra of Rev Response Element (RRE)-IIB were collected in the presence of different concentrations of the peptides
A comparative analysis reveal that the changes in CD signal is more prominent in case of RSG-1.2 binding than that of Rev binding
Summary
The large pre mRNA transcript produced by HIV-1, during its early phase of lifecycle, undergoes alternative splicing to generate small mRNA transcripts for production of certain regulatory proteins. Different biochemical studies have shown that, Rev-RRE interaction is primarily mediated through an arginine rich motif (ARM) of the protein [12,13] which binds to IIB region of RRE in a-helical conformation [14] This interaction brings about conformational changes to the RNA and facilitates binding of up to 8 Rev monomers [15,16,17,18] in a cooperative manner. Important residues of Rev protein involved in Rev-RRE binding have been identified by different in vitro selection, chemical modification and mutagenesis studies [18,26,27,28,29] Based on these studies many groups have tried to generate peptides with increased affinity and specificity to block the Rev-RRE-IIB complex formation. Rev binding causes the flipping of U72 residue towards the aqueous solution [31], whereas RSG-1.2 binding results in flipping of U72 residue inside the major groove region facilitating the stacking of U72 with Arg residue [32]
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