Poster abstracts

Poster number 42 submitted by Michele Tolbert

Biophysical characterization of hnRNPA1 interactions with the Enterovirus 71 IRES

Michele Tolbert (Department of Chemistry, Case Western Reserve University), Jeffrey Levengood (Department of Chemistry, Case Western Reserve University), Mei-Ling Li (Department of Molecular Genetics, Microbiology & Immunology, UMDNJ-Robert Wood Johnson Medical School), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
Enterovirus 71 (EV71), a positive single stranded RNA virus in the picornaviridae family, is one of the two major etiological agents involved in hand, foot and mouth disease. The lack of a 5’ m7G cap results in the utilization of a highly structured six stem loop IRES and various host IRES trans-acting factors (ITAFs) during translational initiation (1). A key ITAF implicated in EV71 IRES activity is heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) which is believed to interact with Stem Loop II (SLII) in the EV71 IRES, making it likely that SLII is critical to IRES dependent activity. To examine the nature of hnRNPA1:SLII interactions, a bioinformatics, NMR and Isothermal Calorimetric (ITC) based approach was utilized. To elucidate characteristic SLII sequence and secondary structure similarities between different EV71 strains, complete genome alignment and SLII secondary structure prediction occurred. All predicted EV71 SLII secondary structures presented a phylogenetically conserved secondary structure. This high degree of secondary and primary sequence conservation in the bulge and hairpin regions make it likely that these regions are critical to hnRNPA1:SLII interactions. 2D NOESY analysis coupled to secondary structure prediction confirmed the presence of the upper six nucleotide hairpin and the lower five nucleotide bulge in SLII. To explore the hnRNPA1 binding nature of these regions, ITC studies on a wildtype SLII and a hairpin only mutant were performed. Wildtype ITC data suggests a propensity for hnRNPA1 to bind to SLII in a sequential manner involving two unique binding sites, i.e. the lower bulge and upper hairpin. To examine this likelihood, a hairpin only mutant was designed to examine binding exclusive to the hairpin; an additional in vivo viral activity assay was also utilized. The high degree of secondary structure conservation coupled to observed hnRNPA1 binding patterns independent of the bulge make it likely that SLII could act as a novel EV71 therapeutic site.

References:
1. Lin, J., et. al. (2009) hnRNP A1 Interacts with the 5’ Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication. Journal of Virology 83: 6106-6114.

Keywords: EV71, hnRNPA1