Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Probing the Molecular Determinants of HIV Alternative Splicing: NMR and Thermodynamic Studies of UP1/ESS3
Alternative splicing of the human immunodeficiency virus type-1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impairs HIV-1 replication (1-2). The cellular protein, hnRNP A1 (A1), regulates splicing activity at several highly conserved 3’ alternative splice sites (ssA2, ssA3, and ssA7) by binding 5’-UAG-3’ motifs embedded within regions containing higher-order RNA structure (3). The biophysical determinants of A1/splice site recognition remain poorly defined in HIV-1; thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the first 3D structure of an HIV-1 splicing regulatory RNA element, exon splicing silencer 3 (ESS3, located at ssA7), has been determined by solution 2H-edited NMR spectroscopy and restrained molecular dynamic simulations (see below). ESS3 adopts a 27-nucleotide hairpin loop where the first 20 base pairs form an A-helical structure. The helix is interrupted by a pH sensitive A+C base pair that is conserved across several HIV-1 isolates. The seven nucleotide hairpin contains the high affinity A1 responsive 5’-UAGU-3’ epitope, and a proximal 5’-GAU-3’ motif. The NMR structure shows that the heptaloop adopts a preformed conformation stabilized by base stacking and non-canonical interactions. Significantly, the apex of the loop is quasi-symmetric where UA dinucleotide steps from the 5’-UAGU-3’ and 5’-GAU-3’ motifs stack on opposite sides of the hairpin - thereby providing a possible A1 binding platform. To further probe the biophysical determinants of high-affinity A1/ESS3 recognition, the thermodynamic profile (ΔG°298K,ΔH°298K,ΔS°298K, and Kd) was measured via ITC for a C-terminal A1 deletion mutant (UP1). UP1 interacts with ESS3 via an enthalpically driven process - giving rise to a complex with nanomolar affinity. The ESS3 binding interface of UP1 was mapped via 15N-1H HSQC titrations. The results show that the UP1/ESS3 binding interface is broad and involves regions not restricted to the beta-pleated sheet. Taken together, we present the first quantitative study of a host factor/HIV-1 splicing RNA element.
This research has been supported by start up funds from Miami University, College of Arts and Sciences.
Blanton S. Tolbert
Department of Chemistry and Biochemistry