Identification of the Functional Roles of Cellular Proteins That Associate with 5’ Untranslated Region

Associated with severe neurological disorders in children, enterovirus 71 (EV71) has been implicated as the etiological agent in several large-scale outbreaks with mortalities. Studies on viral host interaction in EV71-infected cells are important because rational drug discovery is based on the molecular targets that are identified from such studies. This research proposal will focus on how host cellular proteins and the viral 5’ untranslated region (5’ UTR) interact with each other. 5’ UTR in picornaviral genome contains a cloverleaf-like structure that is important for viral RNA synthesis and an internal ribosomal entry site (IRES) that is crucial to mRNA translation. An RNA-affinity chromatography approach using biotinylated RNA was employed to purify and characterize cellular proteins that are associated with EV71 5’ UTR. Multiple proteins were identified that appear to interact specifically with EV71 5’ UTR, some of which have not been reported. In this proposal two novel proteins, heterogeneous nuclear ribonuclear protein K (hnRNP K) and far upstream element binding protein 2 (FBP2) are chosen for further studying. The research proposal attempts to verify the specific interactions between these two proteins and EV71 5’ UTR. hnRNP K was found to bind to the cloverleaf structure specifically and to enhance viral RNA synthesis. The proposed investigation will elucidate the mechanism by which hnRNP K affects viral RNA synthesis. RNA stability assay will be applied to determine whether hnRNP K influences viral RNA half-life and then increases viral RNA synthesis. In vitro RNA replication assay will be also conducted to understand how hnRNP K interacts with other viral or cellular proteins in the replication complex. In addition to having a cloverleaf-like structure, EV71 5’ UTR also contains IRES. FBP2 was verified to bind to IRES and negatively affect viral protein synthesis. We intend to study how FBP2 acts as a negative IRES trans-acting factor (ITAF). Does it cause viral RNA to decay or does it outcompete with other positive ITAFs? The impacts of FBP2 on viral IRES activity, viral translation and viral yields will be evaluated by knocking-down or over-expressing FBP2. The effect of FBP2 on EV71 RNA integrity will be examined to determine whether FBP2 decays viral RNA. Competition assay will be performed to understand whether FBP2 outcompete other positive ITAFs (such as PTB and FBP) such that it acts as a negative regulator. To elucidate the mechanisms by which cloverleaf-associated protein (such as hnRNP K) and IRES-associated protein (such as FBP2) act in viral RNA replication and translation is very significant and important. Understanding how cellular proteins interact with viral RNA and their roles in viral infection would provide insights that would help in designing novel antiviral agents based on those interactions.

Project ID:PC10006-0027
External Project ID:NSC100-3112-B182-002