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Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway.

ABSTRACT: The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication.

SUBMITTER: Srinivasakumar N

PROVIDER: S-EPMC3229575 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway.

Srinivasakumar Narasimhachar N

PloS one 20111202 12

The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T- ...[more]

PMID: 22164294

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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

Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway.

Publications

Rev-free HIV-1 gene delivery system for targeting Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway.

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