Not listed DEFINING THE RNA-BINDING LANDSCAPE OF FLAVIVIRUS CAPSID PROTEIN - PROJECT SUMMARY/ABSTRACT WITHIN THE FLAVIVIRIDAE FAMILY OF VIRUSES, THE GENUS ORTHOFLAVIVIRUS IS PRIMARILY COMPRISED OF TICK AND MOSQUITO- BORNE VECTORS THAT ARE ENDEMIC TO MANY REGIONS OF THE WORLD. DENGUE VIRUS (DENV) AND ZIKA VIRUS (ZIKV) ARE TWO OF THE MOST PROMINENT FLAVIVIRUSES, BOTH OF WHICH POSE A SUBSTANTIAL INFECTIOUS HEALTH RISK AND LACK EFFECTIVE THERAPEUTIC INTERVENTIONS. DENV AND ZIKV POSSESS AN ~11 KB RNA GENOME THAT ENCODES A SINGLE POLYPROTEIN WHICH IS TRANSLATED, TRANSLOCATED, AND PROTEOLYTICALLY PROCESSED ON THE HOST ENDOPLASMIC RETICULUM (ER) MEMBRANE, COINCIDENT WITH A REMODELING OF HOST CELL ER-ASSOCIATED TRANSLATION. THE FIRST PROTEIN TRANSLATED FROM THE OPEN READING FRAME OF ALL FLAVIVIRUS GENOMIC RNAS (GRNAS) IS CAPSID (C), A 12 KDA RNA BINDING PROTEIN FUNCTIONING IN GRNA PACKAGING. HOWEVER, C LACKS KNOWN RNA-BINDING MOTIFS AND THE BASIS OF SPECIFICITY FOR GRNA BINDING AS WELL AS THE POTENTIAL FOR C TO BIND HOST RNAS REMAINS TO BE ESTABLISHED. GIVEN PRELIMINARY DATA THAT DENV AND ZIKV C BINDS TO HOST RNAS IN CELLS, THE WORK PROPOSED HERE WILL TEST THE HYPOTHESIS THAT FLAVIVIRUS C-RNA INTERACTOMES EXTEND BEYOND GRNA TO IMPACT HOST RNA METABOLISM THROUGH MODULATION OF TRANSLATION OR STABILITY OF INTERACTING RNAS. TWO SPECIFIC AIMS ARE PROPOSED. IN AIM 1, C BINDING TO HOST RNAS IN ZIKV AND DENV CELL INFECTION MODELS WILL BE INVESTIGATED. CELL FRACTIONATION COUPLED WITH ENHANCED RNA-INTERACTOME CAPTURE (ERIC) WILL BE EMPLOYED TO IDENTIFY THE DYNAMIC HOST POLY(A) MRNA- BOUND PROTEOME DURING THE VIRAL LIFECYCLE. ADDITIONALLY, A RECENT TECHNIQUE DEVELOPED IN OUR LABORATORY, LEAP- RBP, WILL BE EMPLOYED FOR SELECTIVE ISOLATION OF TOTAL UV-CROSSLINKED RNA-PROTEIN COMPLEXES FOLLOWED BY IMMUNOAFFINITY PURIFICATION OF C AND ENHANCED CLIP-SEQ (ECLIP) ANALYSIS TO DETERMINE THE COMPREHENSIVE C- BOUND TRANSCRIPTOME AND TO IDENTIFY CANDIDATE C BINDING MOTIFS. IN AIM 2 THE MECHANISTIC IMPACT OF C BINDING ON HOST RNA METABOLISM WILL BE INVESTIGATED USING INDUCIBLE ECTOPIC EXPRESSION OF DENV AND ZIKV C IN HUMAN CELLS. THE IMPACT OF C EXPRESSION ON RNA STABILITY WILL BE EXAMINED THROUGH TOTAL RNA-SEQ AND ACTINOMYCIN-D TRANSCRIPTIONAL INHIBITION TO QUANTIFY CHANGES IN RNA HALF-LIFE. CHANGES IN MRNA TRANSLATION WILL BE ASSESSED BY CELL FRACTIONATION AND RIBO-SEQ ANALYSIS AND ORTHOGONALLY VALIDATED BY MASS SPECTROMETRIC ANALYSIS OF PUROMYCIN-TAGGED RIBOSOME-ASSOCIATED NASCENT CHAINS (PUNCH-P). THE PROPOSED AIMS ARE EXPECTED TO FILL GAPS IN OUR UNDERSTANDING OF FLAVIVIRUS CAPSID PROTEIN BIOLOGY THROUGH DISCOVERY OF C-HOST CELL RNA INTERACTIONS AND ELUCIDATION OF HOW THESE INTERACTIONS MAY FACILITATE VIRAL PATHOGENICITY. THE PROPOSED RESEARCH WILL BE CONDUCTED IN COLLABORATIVE VIROLOGY AND RNA BIOLOGY LABORATORIES WITH ACCESS TO CUTTING-EDGE SEQUENCING AND PROTEOMICS TECHNOLOGIES. THE TRAINING PLAN INCLUDES RIGOROUS MENTORSHIP, COURSEWORK, AND PROFESSIONAL DEVELOPMENT ACTIVITIES TAILORED FOR PREPARATION FOR AN INDEPENDENT RESEARCH CAREER. $50.1k 7/17/26 Not listed DEFINING THE RNA-BINDING LANDSCAPE OF FLAVIVIRUS CAPSID PROTEIN - PROJECT SUMMARY/ABSTRACT WITHIN THE FLAVIVIRIDAE FAMILY OF VIRUSES, THE GENUS ORTHOFLAVIVIRUS IS PRIMARILY COMPRISED OF TICK AND MOSQUITO- BORNE VECTORS THAT ARE ENDEMIC TO MANY REGIONS OF THE WORLD. DENGUE VIRUS (DENV) AND ZIKA VIRUS (ZIKV) ARE TWO OF THE MOST PROMINENT FLAVIVIRUSES, BOTH OF WHICH POSE A SUBSTANTIAL INFECTIOUS HEALTH RISK AND LACK EFFECTIVE THERAPEUTIC INTERVENTIONS. DENV AND ZIKV POSSESS AN ~11 KB RNA GENOME THAT ENCODES A SINGLE POLYPROTEIN WHICH IS TRANSLATED, TRANSLOCATED, AND PROTEOLYTICALLY PROCESSED ON THE HOST ENDOPLASMIC RETICULUM (ER) MEMBRANE, COINCIDENT WITH A REMODELING OF HOST CELL ER-ASSOCIATED TRANSLATION. THE FIRST PROTEIN TRANSLATED FROM THE OPEN READING FRAME OF ALL FLAVIVIRUS GENOMIC RNAS (GRNAS) IS CAPSID (C), A 12 KDA RNA BINDING PROTEIN FUNCTIONING IN GRNA PACKAGING. HOWEVER, C LACKS KNOWN RNA-BINDING MOTIFS AND THE BASIS OF SPECIFICITY FOR GRNA BINDING AS WELL AS THE POTENTIAL FOR C TO BIND HOST RNAS REMAINS TO BE ESTABLISHED. GIVEN PRELIMINARY DATA THAT DENV AND ZIKV C BINDS TO HOST RNAS IN CELLS, THE WORK PROPOSED HERE WILL TEST THE HYPOTHESIS THAT FLAVIVIRUS C-RNA INTERACTOMES EXTEND BEYOND GRNA TO IMPACT HOST RNA METABOLISM THROUGH MODULATION OF TRANSLATION OR STABILITY OF INTERACTING RNAS. TWO SPECIFIC AIMS ARE PROPOSED. IN AIM 1, C BINDING TO HOST RNAS IN ZIKV AND DENV CELL INFECTION MODELS WILL BE INVESTIGATED. CELL FRACTIONATION COUPLED WITH ENHANCED RNA-INTERACTOME CAPTURE (ERIC) WILL BE EMPLOYED TO IDENTIFY THE DYNAMIC HOST POLY(A) MRNA- BOUND PROTEOME DURING THE VIRAL LIFECYCLE. ADDITIONALLY, A RECENT TECHNIQUE DEVELOPED IN OUR LABORATORY, LEAP- RBP, WILL BE EMPLOYED FOR SELECTIVE ISOLATION OF TOTAL UV-CROSSLINKED RNA-PROTEIN COMPLEXES FOLLOWED BY IMMUNOAFFINITY PURIFICATION OF C AND ENHANCED CLIP-SEQ (ECLIP) ANALYSIS TO DETERMINE THE COMPREHENSIVE C- BOUND TRANSCRIPTOME AND TO IDENTIFY CANDIDATE C BINDING MOTIFS. IN AIM 2 THE MECHANISTIC IMPACT OF C BINDING ON HOST RNA METABOLISM WILL BE INVESTIGATED USING INDUCIBLE ECTOPIC EXPRESSION OF DENV AND ZIKV C IN HUMAN CELLS. THE IMPACT OF C EXPRESSION ON RNA STABILITY WILL BE EXAMINED THROUGH TOTAL RNA-SEQ AND ACTINOMYCIN-D TRANSCRIPTIONAL INHIBITION TO QUANTIFY CHANGES IN RNA HALF-LIFE. CHANGES IN MRNA TRANSLATION WILL BE ASSESSED BY CELL FRACTIONATION AND RIBO-SEQ ANALYSIS AND ORTHOGONALLY VALIDATED BY MASS SPECTROMETRIC ANALYSIS OF PUROMYCIN-TAGGED RIBOSOME-ASSOCIATED NASCENT CHAINS (PUNCH-P). THE PROPOSED AIMS ARE EXPECTED TO FILL GAPS IN OUR UNDERSTANDING OF FLAVIVIRUS CAPSID PROTEIN BIOLOGY THROUGH DISCOVERY OF C-HOST CELL RNA INTERACTIONS AND ELUCIDATION OF HOW THESE INTERACTIONS MAY FACILITATE VIRAL PATHOGENICITY. THE PROPOSED RESEARCH WILL BE CONDUCTED IN COLLABORATIVE VIROLOGY AND RNA BIOLOGY LABORATORIES WITH ACCESS TO CUTTING-EDGE SEQUENCING AND PROTEOMICS TECHNOLOGIES. THE TRAINING PLAN INCLUDES RIGOROUS MENTORSHIP, COURSEWORK, AND PROFESSIONAL DEVELOPMENT ACTIVITIES TAILORED FOR PREPARATION FOR AN INDEPENDENT RESEARCH CAREER. $0 7/17/26 Not listed DEFINING THE RNA-BINDING LANDSCAPE OF FLAVIVIRUS CAPSID PROTEIN - PROJECT SUMMARY/ABSTRACT WITHIN THE FLAVIVIRIDAE FAMILY OF VIRUSES, THE GENUS ORTHOFLAVIVIRUS IS PRIMARILY COMPRISED OF TICK AND MOSQUITO- BORNE VECTORS THAT ARE ENDEMIC TO MANY REGIONS OF THE WORLD. DENGUE VIRUS (DENV) AND ZIKA VIRUS (ZIKV) ARE TWO OF THE MOST PROMINENT FLAVIVIRUSES, BOTH OF WHICH POSE A SUBSTANTIAL INFECTIOUS HEALTH RISK AND LACK EFFECTIVE THERAPEUTIC INTERVENTIONS. DENV AND ZIKV POSSESS AN ~11 KB RNA GENOME THAT ENCODES A SINGLE POLYPROTEIN WHICH IS TRANSLATED, TRANSLOCATED, AND PROTEOLYTICALLY PROCESSED ON THE HOST ENDOPLASMIC RETICULUM (ER) MEMBRANE, COINCIDENT WITH A REMODELING OF HOST CELL ER-ASSOCIATED TRANSLATION. THE FIRST PROTEIN TRANSLATED FROM THE OPEN READING FRAME OF ALL FLAVIVIRUS GENOMIC RNAS (GRNAS) IS CAPSID (C), A 12 KDA RNA BINDING PROTEIN FUNCTIONING IN GRNA PACKAGING. HOWEVER, C LACKS KNOWN RNA-BINDING MOTIFS AND THE BASIS OF SPECIFICITY FOR GRNA BINDING AS WELL AS THE POTENTIAL FOR C TO BIND HOST RNAS REMAINS TO BE ESTABLISHED. GIVEN PRELIMINARY DATA THAT DENV AND ZIKV C BINDS TO HOST RNAS IN CELLS, THE WORK PROPOSED HERE WILL TEST THE HYPOTHESIS THAT FLAVIVIRUS C-RNA INTERACTOMES EXTEND BEYOND GRNA TO IMPACT HOST RNA METABOLISM THROUGH MODULATION OF TRANSLATION OR STABILITY OF INTERACTING RNAS. TWO SPECIFIC AIMS ARE PROPOSED. IN AIM 1, C BINDING TO HOST RNAS IN ZIKV AND DENV CELL INFECTION MODELS WILL BE INVESTIGATED. CELL FRACTIONATION COUPLED WITH ENHANCED RNA-INTERACTOME CAPTURE (ERIC) WILL BE EMPLOYED TO IDENTIFY THE DYNAMIC HOST POLY(A) MRNA- BOUND PROTEOME DURING THE VIRAL LIFECYCLE. ADDITIONALLY, A RECENT TECHNIQUE DEVELOPED IN OUR LABORATORY, LEAP- RBP, WILL BE EMPLOYED FOR SELECTIVE ISOLATION OF TOTAL UV-CROSSLINKED RNA-PROTEIN COMPLEXES FOLLOWED BY IMMUNOAFFINITY PURIFICATION OF C AND ENHANCED CLIP-SEQ (ECLIP) ANALYSIS TO DETERMINE THE COMPREHENSIVE C- BOUND TRANSCRIPTOME AND TO IDENTIFY CANDIDATE C BINDING MOTIFS. IN AIM 2 THE MECHANISTIC IMPACT OF C BINDING ON HOST RNA METABOLISM WILL BE INVESTIGATED USING INDUCIBLE ECTOPIC EXPRESSION OF DENV AND ZIKV C IN HUMAN CELLS. THE IMPACT OF C EXPRESSION ON RNA STABILITY WILL BE EXAMINED THROUGH TOTAL RNA-SEQ AND ACTINOMYCIN-D TRANSCRIPTIONAL INHIBITION TO QUANTIFY CHANGES IN RNA HALF-LIFE. CHANGES IN MRNA TRANSLATION WILL BE ASSESSED BY CELL FRACTIONATION AND RIBO-SEQ ANALYSIS AND ORTHOGONALLY VALIDATED BY MASS SPECTROMETRIC ANALYSIS OF PUROMYCIN-TAGGED RIBOSOME-ASSOCIATED NASCENT CHAINS (PUNCH-P). THE PROPOSED AIMS ARE EXPECTED TO FILL GAPS IN OUR UNDERSTANDING OF FLAVIVIRUS CAPSID PROTEIN BIOLOGY THROUGH DISCOVERY OF C-HOST CELL RNA INTERACTIONS AND ELUCIDATION OF HOW THESE INTERACTIONS MAY FACILITATE VIRAL PATHOGENICITY. THE PROPOSED RESEARCH WILL BE CONDUCTED IN COLLABORATIVE VIROLOGY AND RNA BIOLOGY LABORATORIES WITH ACCESS TO CUTTING-EDGE SEQUENCING AND PROTEOMICS TECHNOLOGIES. THE TRAINING PLAN INCLUDES RIGOROUS MENTORSHIP, COURSEWORK, AND PROFESSIONAL DEVELOPMENT ACTIVITIES TAILORED FOR PREPARATION FOR AN INDEPENDENT RESEARCH CAREER. $50.1k 7/17/26