The overall goal of this program component is to elucidate the mechanism by which HIV Rev binds to the Rev Response Element and then assembles an oligomeric ribonucleoprotein (RNP) complex that mediates nuclear export of the viral mRNA. In parallel with the mechanistic studies, we will develop novel fluorescence-based assays that will be used to screen compound libraries for inhibitors of the RNA-binding and oligomerization functions of Rev. We will also perform biophysical studies of compounds identified in these screens to elucidate their mechanism of action. This project will lead to a detailed understanding of the kinetic pathway and cooperativity of oligomeric RNP complex assembly. In addition to providing information on basic Rev function, an understanding of the assembly mechanism will contribute to efforts to create small molecules that disrupt proper assembly of the RNP compex and thereby prevent export of the viral mRNA from the nucleus.
The Specific Aims are: (1) Elucidate the mechanism of multimeric Rev assembly on the RRE. (2) Characterize RNA conformational changes induced during multimeric Rev-RRE complex assembly. (3) Develop fluorescence-based screening assays to identify small molecules that interfere with Rev-RRE binding or Rev-Rev multimerization. (4) Establish the mechanism of inhibition of small molecules selected from the fluorescence-based screens. A novel single-molecule fluorescence imaging technique will be used to observe spontaneous assembly of multimeric Rev complexes on a critical fragment of the RRE, revealing the sequence of events and the molecular basis for cooperative assembly. Fluorescence resonance energy transfer (FRET) methods will be used to probe the structure of the RRE fragment at each stage of complex assembly, in both ensemble and single-molecule experiments. These spectroscopic methods will be adapted for use in screening compound libraries generated in other program components, allowing for specific selection of molecules that disrupt the initial binding of Rev to the RRE or the subsequent oligomerization steps. This project will support the drug discovery effort and help to identify interesting inhibitor-target complexes for structural characterization in other program components.
| 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 |
