HIV-1 Rev expressed in recombinant Escherichia coli: purification, polymerization, and conformational properties

Abstract

The high-level expression of HIV-1 Rev in Escherichia coli is described. Protein in crude bacterial extracts was dissociated from bound nucleic acid with urea. A simple purification and renaturation protocol, monitored by circular dichroism, is described which results in high yields of pure protein. The purified protein binds with high affinity to the Rev-responsive element mRNA and has nativelike spectroscopic properties. The protein exhibits concentration-dependent self-association as judged by analytical ultracentrifugation and gel filtration measurements. Purified Rev showed reversible heat-induced aggregation over the temperature range 0-30 degrees C. This hydrophobic-driven and nonspecific protein association was inhibited by low concentrations of sulfate ions. Rev solutions at greater than 80 micrograms/mL, incubated at 0-4 degrees C, slowly polymerized to form long hollow fibers of 20-nm diameter. Filament formation occurs at a lower protein concentration and more rapidly in the presence of Rev-responsive mRNA. The nucleic acid containing filaments are about 8 nm in diameter and up to 0.4 micron in length. On the basis of physical properties of the purified protein, we have suggested that in the nucleus of infected cells, Rev binding to the Rev-responsive region of env mRNA may be followed by helical polymerization of the protein which results in coating of the nucleic acid. Coated nucleic acid could be protected from splicing in the nucleus and exported to the cytoplasm.

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

“…Having these synthesis tools in hand, we plan to study the effect of post-translational modifications [54][55][56] on the function and structure of the Rev protein, along with introducing chemical switches [57,58] to control Rev folding and self-assembly, to better understand these processes on the function of Rev protein. [4,53,59] …”

“…The arginine-rich motif (ARM, residues 34-50) acts as the nuclear-localization signal (NLS) and as a binding site for the Rev response element (RRE), which is present in all incompletely spliced viral mRNAs. [3][4][5][6] Although Rev was discovered nearly three decades ago, [7] biochemical and structural studies aimed at understanding the structural and functional aspects of the Rev protein are still limited because of its strong tendency to oligomerize, aggregate, and precipitate. [4,8,9] As a result, the majority of the Rev studies have been performed on peptides derived from the protein sequence, and on Rev conjugates such as Rev-GFP.…”

“…[3][4][5][6] Although Rev was discovered nearly three decades ago, [7] biochemical and structural studies aimed at understanding the structural and functional aspects of the Rev protein are still limited because of its strong tendency to oligomerize, aggregate, and precipitate. [4,8,9] As a result, the majority of the Rev studies have been performed on peptides derived from the protein sequence, and on Rev conjugates such as Rev-GFP. [6,[10][11][12] Only very recently was the X-ray structure of this protein solved; however, it was complexed with a specifically engineered monoclonal Fab (fragment antigen-binding) antibody that served as the solubilizing agent.…”

Structural and Functional Characterization of RNA-binding Site of Rev and Rev-Response-Element RNA Complex via All Atom Molecular Dynamics Simulations

“…Rev is a good target for antiviral therapies, since Rev is absolutely necessary for HIV-1 replication [27,28]. It has been shown that various organic compounds have the ability to target the Rev/RRE interaction [29].…”

“…It has been shown that various organic compounds have the ability to target the Rev/RRE interaction [29]. Neomycin B, diphenylfuran cation, and proflavine are small molecules that can prevent Rev from binding to the RRE sequence [27,29,30]. If Rev is incapable of binding to the RRE on the pre-mRNA, the RNA will not be exported to the cytoplasm, also resulting in lack of necessary structural proteins.…”