Dynamic regulation of RNA in the Zika virus life cycle

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RNA is arguably the most versatile biological macromolecule owing to its ability to both encode genetic information and to fold back on itself to form complex three-dimensional structures capable of highly specific ligand recognition and catalysis. RNA structure is also dynamic, meaning that a structure that plays an important role in one biological state may not persist under different biological conditions. Thus, studying RNA structure and organization is crucial to understanding its biological function. RNA secondary and higher ordered structures are also known to play a fundamental role in the life cycle of RNA viruses. Specific viral RNA structures have been demonstrated to act in a cell-type and species-specific manner to regulate the life cycle of mosquito-borne RNA viruses, such as Zika virus (ZIKV). We hypothesize that the ZIKV genomic RNA forms multiple dynamic structures that are critical for the viral life cycle. To test this hypothesis, we will perform Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) analysis on ZIKV RNAs in several biological contexts to identify RNA structures important for the viral life cycle. The following specific aims will be explored: 1. Characterization of the ZIKV genomic RNA in viral particles. To date, no viral packaging signals have been identified for ZIKV. In this aim, we will combine in vitro and in virio SHAPE analysis with photo-activatable cross-linking (PAR-CL). We anticipate that this will help define packaging signals and reveal the complex structure of the ZIKV genome inside viral particles. 2. Characterization of the dynamic structure of the ZIKV genomic RNA throughout the life cycle in human and mosquito cells. We will perform SHAPE analysis on viral RNAs stalled in one or more stages of the viral life cycle to generate a picture of the dynamics of RNA structure throughout the life cycle in human and mosquito cells. Identified regulatory motifs will be subject to mutagenesis and effects on viral translation, replication, and particle production will be assessed as well as the differential requirements between hosts. 3. Characterization of the negative-strand intermediate. To date, no studies have explored the structure of the negative-strand RNA intermediate. To identify the RNA structures important for positive-strand RNA synthesis, we will combine RNA structure prediction with SHAPE analysis of the negative-strand RNA to reveal insight into the requirements for positive-strand RNA synthesis. The proposed research is highly significant as it will improve our understanding of ZIKV biology. Characterization of novel RNA regulatory motifs will allow us to define new regulatory elements and refine models for viral translation, replication, and packaging. Moreover, identifying differential requirements in human vs. mosquito cells will provide insight into ZIKV evolution, adaptation, and epidemiological fitness.