Project Grant F31AI189061

CHARACTERIZING THE STRUCTURE AND FUNCTION OF A VIRAL RNA ELEMENT IN INHIBITING AN ANTIVIRAL PROTEIN - PROJECT SUMMARY RNA VIRUSES ARE A CONSTANT THREAT TO GLOBAL HEALTH AND UNDERSTANDING THEIR INTERACTIONS WITH HOST CELLS AT THE MOLECULAR LEVEL IS NECESSARY FOR IDENTIFYING THERAPEUTIC TARGETS. VIRUSES CONTAIN DIVERSE TYPES OF STRUCTURED RNA ELEMENTS WITHIN THEIR GENOMES WITH A VARIETY OF FUNCTIONS THAT ARE CRITICAL FOR SUCCESSFUL INFECTION. ONE SUCH STRUCTURED RNA ELEMENT WAS IDENTIFIED WITHIN POLIOVIRUS AND PROTECTS THE GENOMIC RNA FROM DEGRADATION BY THE MAMMALIAN ENDONUCLEASE, RIBONUCLEASE L (RNASE L). THIS RNA WAS FOUND TO BE A COMPETITIVE INHIBITOR OF RNASE L, PREVENTING DEGRADATION OF VIRAL GENOMIC RNA. PREVIOUS EXPERIMENTS HAVE CONFIRMED THE SECONDARY STRUCTURE OF THE POLIOVIRUS COMPETITIVE INHIBITOR RNA (CIRNA) AS WELL AS KEY TERTIARY CONTACTS REQUIRED FOR INHIBITION FUNCTION; HOWEVER, THE 3D STRUCTURE OF THE POLIOVIRUS CIRNA AND ITS SPECIFIC INTERMOLECULAR INTERACTIONS WITH RNASE L THAT ENABLE INHIBITION REMAIN UNRESOLVED. ADDITIONALLY, THE CIRNA ELEMENT WAS IDENTIFIED IN THE POLIOVIRUS GENOME BUT ITS PRESENCE IN DIVERGENT RNA VIRUSES HAS NOT BEEN EXPLORED OR CLASSIFIED. I HYPOTHESIZE THE CIRNA STRUCTURE DIRECTLY INHIBITS RNASE L BY PHYSICALLY BLOCKING ITS ACTIVE SITE. THE CIRNA RELIES ON CRUCIAL SECONDARY AND TERTIARY CONTACTS TO MAINTAIN ITS INHIBITORY PROPERTIES. I WILL IDENTIFY CIRNA ELEMENTS IN ADDITIONAL VIRAL GENOMES AND DEFINE THE PHYLOGENETIC DISTRIBUTION OF THIS CLASS OF RNAS. TO TEST THESE HYPOTHESES, I WILL USE A COMBINATION OF BIOPHYSICAL TECHNIQUES, STRUCTURAL BIOLOGY, BIOCHEMICAL ASSAYS, AND BIOINFORMATICS. AIM 1. I WILL USE A BIOINFORMATIC APPROACH TO QUERY A VIRAL GENOMIC DATABASE FOR CIRNA ELEMENTS AND TEST NEW PUTATIVE HITS FOR RNASE L INHIBITION ACTIVITY USING A FLUORESCENCE-BASED ASSAY. I WILL USE BIOCHEMICAL METHODS TO MAP THE BINDING SITE OF RNASE L ON CIRNA TO FIND THE CONSERVED BINDING SITE AMONG ALL EXAMPLES. AIM 2. I WILL CHARACTERIZE THE INTERACTION BETWEEN THE CIRNA AND RNASE L (AND THEIR MUTANTS) USING MICROSCALE THERMOPHORESIS TO QUANTIFY THE BINDING AFFINITY AND MEASURE THE STOICHIOMETRY BETWEEN THESE TWO MOLECULES AND BIOCHEMICALLY RELEVANT MUTANTS. TO FULLY CHARACTERIZE THIS INTERACTION ON A MOLECULAR LEVEL, I WILL USE CRYO-ELECTRON MICROSCOPY TO SOLVE THE STRUCTURE OF THE CIRNA-RNASE L COMPLEX. THE WORK OUTLINED IN THIS PROPOSAL WILL PROVIDE INSIGHT INTO HOW STRUCTURED RNA ELEMENTS DIRECTLY INTERACT WITH HOST PROTEIN FACTORS, AND HOW THESE HAVE EVOLVED IN THE VIRAL WORLD.