Replication of the human immunodeficiency virus type-1 (HIV-1) in the nucleus requires the virus-encoded regulatory protein Rev. Because of its key role in the viral life cycle, Rev is an attractive therapeutic target for small molecules. Rev binds to a structured RNA element, termed the Rev Response Element (RRE), which is found in the unspliced and singly spliced HIV mRNAs that encode the Gag, Pol and Env proteins. The binding and multimerization of Rev at the RRE recruits the nuclear export receptor Crm1, and as a consequence induces export of the HIV mRNAs to the cytoplasm to allow their translation. It is known that Crm1 and the small GTPase Ran are involved in Rev-mediated viral mRNA export, and a number of other potential export factors have been described as well. However, the composition and assembly of the nuclear export complex based on the Rev/RRE interaction remain poorly understood. The goals of this project are to investigate these issues in detail, using a combination of biochemical and functional approaches.
The specific aims are: 1) Proteomic and in vitro binding assays will be used to analyze the nuclear export complex formed on the Rev oligomer associated with the RRE; 2) A permeabilized cell assay will be used to analyze the soluble factors involved in nuclear export of an HIV-derived mRNA containing the RRE; 3) In vivo functional studies will be carried out to analyze potential factors involved in mRNA export mediated by the Rev-RRE complex. The other investigators in this program will study in detail the Rev-RRE interaction and Rev multimerization, and will attempt to identify small molecule inhibitors of these associations. In turn, we will test promising compounds for their effect on HIV mRNA export in cells. This work is expected to lead to a greater understanding of how Rev mediates HIV mRNA export, and could help to identify promising lead compounds for drug development.
|Marenchino, Marco; Armbruster, David W; Hennig, Mirko (2009) Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev. Protein Expr Purif 63:112-9|
|Pond, Stephanie J K; Ridgeway, William K; Robertson, Rae et al. (2009) HIV-1 Rev protein assembles on viral RNA one molecule at a time. Proc Natl Acad Sci U S A 106:1404-8|
|Carlomagno, Teresa; Amata, Irene; Williamson, James R et al. (2008) NMR assignments of HIV-2 TAR RNA. Biomol NMR Assign 2:167-9|
|Pljevaljcic, Goran; Millar, David P (2008) Single-molecule fluorescence methods for the analysis of RNA folding and ribonucleoprotein assembly. Methods Enzymol 450:233-52|
|Edgcomb, Stephen P; Aschrafi, Angelique; Kompfner, Elizabeth et al. (2008) Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes. Protein Sci 17:420-30|
|Scott, Lincoln G; Hennig, Mirko (2008) RNA structure determination by NMR. Methods Mol Biol 452:29-61|
|Hennig, Mirko; Scott, Lincoln G; Sperling, Edit et al. (2007) Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J Am Chem Soc 129:14911-21|
|Hennig, Mirko; Munzarova, Marketa L; Bermel, Wolfgang et al. (2006) Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA. J Am Chem Soc 128:5851-8|