Defining the Structural Mechanisms of RNP Complexes that Regulate Enterovirus Translation

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Project abstract: Enteroviruses (EVs) coordinate protein synthesis and genome replication through their 5'UTR, which is predicted to fold into six stem loops. Stem loop (SL) I facilitates viral genome replication; SLII- VI function as the basic IRES unit for cap-independent translation. Several host RNA binding proteins (referred to as ITAFs), are recruited to the IRES for efficient viral protein synthesis. The current dogma supports a model wherein ITAFs either stabilize or destabilize IRES structure in a conformation that controls ribosome entry. The fundamental knowledge gap is an understanding of mechanisms by which ITAFs functionally interact with the 5'UTR to organize IRES structure. This knowledge is critical to reveal underlying mechanisms by which EVs redirect host factors to maintain infection. The long-term goal is to better understand the molecular mecha- nisms by which EVs subvert host factors to regulate viral gene expression and replication. The overall objec- tive of this proposal is to illuminate how ITAFs functionally interact with the viral IRES to control translation. EV71 and the ITAFs hnRNP A1 and AUF1 serve as excellent models as they bind the same IRES domain SLII to antagonistically fine-tune EV71 translation levels. Moreover, vsRNA1, a virus-derived, small RNA produced by Dicer cleavage of the SLII IRES region, further represses EV71 translation. Our central hypothesis is that conserved viral RNA elements fine-tune EV71 translation levels by assembling unique and antagonistic ribo- nucleoprotein (RNP) complexes, and vsRNA1 compete these interactions to permit more stringent control. The rationale for this research is that ITAF-IRES interactions are central to the synthesis of every EV71 protein and its replication. This new knowledge will thus prove applicable to similar EVs as well. Strong preliminary data lead to three specific aims: (1) Determine the structures of IRES elements that contribute to viral replication; (2) reveal the detailed molecular interactions of antagonistic ITAFs that coordinate viral translation; and (3) identify mechanisms by which viral small RNAs inhibit translation. For Aim1, phylogenetics, chemical probing, NMR, SAXS, and cellular assays will define IRES structural elements that contribute to EV71 translation. For Aim 2, structural, biophysical, and virological assays will reveal protein and RNA conformational changes, and func- tional changes, upon binding of hnRNP A1 and AUF1 to the IRES SLII region, which is critical to translational control. For Aim 3, structural, biochemical, and virological assays will reveal the structural and functional ef- fects of vsRNA1 on antagonistic hnRNP A1- and AUF1-RNPs. The proposal is innovative by employing an in- tegrated program combining structural biochemistry, biophysics, and virological studies that promise to provide significant breakthroughs to better understand viral-host interactions that contribute to EV71 pathogenesis. The research is significant since it promises to deliver unprecedented insights into RNPs and RNA-RNA interac- tions that contribute to EV pathogenesis. This will in turn provide a foundation for identification of new antivirals against EV71 and EV68, which the NIH has recently designated as emerging pathogens.