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Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA.

ABSTRACT: To ensure successful replication, HIV-1 has developed a Rev-mediated RNA transport system that promotes the export of unspliced genomic RNA from nuclei to cytoplasm. This process requires the Rev responsive element (RRE) that is positioned in the viral transcript encoding Env protein, as well as in unspliced and singly spliced viral transcripts. We identified Puralpha, a single-stranded nucleic acid binding protein as a cellular partner for Rev that augments the appearance of unspliced viral RNAs in the cytoplasm. A decrease in the level of Puralpha expression by siRNA diminishes the level of Rev-dependent expression of viral RNA. Through its nucleic acid binding domain, Puralpha exhibits the ability to interact with the multimerization and RBD domains of Rev. Similar to Rev, Puralpha associates with RRE and in the presence of Rev forms a complex with slower electrophoretic mobility than those from Rev:RRE and Puralpha:RRE. The interaction of Puralpha with RRE occurs in the cytoplasm where enhanced association of Rev with RRE is observed. Our data indicate that the partnership of Puralpha with Rev is beneficial for Rev-mediated expression of the HIV-1 genome.

SUBMITTER: Kaminski R

PROVIDER: S-EPMC2575347 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA.

Kaminski Rafal R Darbinian Nune N Sawaya Bassel E BE Slonina Dorota D Amini Shohreh S Johnson Edward M EM Rappaport Jay J Khalili Kamel K Darbinyan Armine A

Journal of cellular biochemistry 20080301 4

To ensure successful replication, HIV-1 has developed a Rev-mediated RNA transport system that promotes the export of unspliced genomic RNA from nuclei to cytoplasm. This process requires the Rev responsive element (RRE) that is positioned in the viral transcript encoding Env protein, as well as in unspliced and singly spliced viral transcripts. We identified Puralpha, a single-stranded nucleic acid binding protein as a cellular partner for Rev that augments the appearance of unspliced viral RNA ...[more]

PMID: 17722108

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Project description:A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/hydroxyl radical) = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.

Dataset Information

Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA.

Publications

Puralpha as a cellular co-factor of Rev/RRE-mediated expression of HIV-1 intron-containing mRNA.

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