Project Grant F32AI186388

FUNCTIONAL SIGNIFICANCE AND MECHANISMS OF EBOLA VIRUS VP24-HOST PROTEIN INTERACTIONS - PROJECT SUMMARY EBOLA VIRUS (EBOV) IS A MEMBER OF THE FILOVIRIDAE FAMILY OF NON-SEGMENTED, NEGATIVE SENSE RNA VIRUSES KNOWN TO CAUSE SPORADIC AND DEADLY OUTBREAKS. AMONG THE EIGHT MAJOR TRANSLATION PRODUCTS ENCODED IN THE FILOVIRUS GENOME, THE VIRAL PROTEIN 24 (VP24) IS CRUCIAL FOR THE PRODUCTION OF INFECTIOUS VIRAL PARTICLES, VIRAL NUCLEOCAPSID ASSEMBLY, AND THE SUPPRESSION OF IFN SIGNALING. TO SUPPRESS IFN SIGNALING, VP24 INTERACTS WITH HOST IMPORTIN- ALPHA (IMPA) NUCLEAR TRANSPORT PROTEINS TO COMPETITIVELY INHIBIT STAT1 INTERACTION WITH IMPA, PREVENTING STAT1 NUCLEAR IMPORT AND THE INDUCTION OF STAT1 DEPENDENT GENE EXPRESSION. WHILE THE VP24-IMPA INTERACTION IS WELL CHARACTERIZED IN THE SUPPRESSION OF IFN SIGNALING, LESS IS KNOWN ABOUT WHETHER THIS HAS ADDITIONAL FUNCTIONAL SIGNIFICANCE. FURTHER, LESS IS KNOWN ABOUT THE IMPORTANCE OF VP24 INTERACTION WITH OTHER HOST PROTEINS. IN A PRELIMINARY AFFINITY PURIFICATION-MASS SPECTROMETRY (AP-MS) PROTEOMIC SCREEN, WE DEMONSTRATED THAT SEVERAL HOST PROTEINS IDENTIFIED AS WILDTYPE VP24 INTERACTORS WERE NOT IDENTIFIED AS INTERACTORS FOR VP24 IMPA-BINDING MUTANTS. HOST PROTEINS THAT WERE IDENTIFIED AS INTERACTORS FOR WILDTYPE BUT NOT MUTANT VP24 MAY USE IMPA TO BRIDGE INTERACTION WITH VP24. IN CONTRAST, HOST PROTEINS THAT INTERACT WITH BOTH WILDTYPE AND IMPA BINDING MUTANT VP24S ARE POTENTIAL DIRECT VP24 INTERACTORS. THEREFORE, I HYPOTHESIZE THAT VP24 CAN INTERACT WITH HOST PROTEINS THROUGH IMPA-INDEPENDENT AND DEPENDENT BINDING MODES, WITH IMPA-BINDING ACTING AS A MECHANISM TO EXPAND THE RANGE OF HOST PROTEINS TARGETED BY VP24. I ALSO HYPOTHESIZE THAT BOTH MODES OF INTERACTION ARE FUNCTIONALLY SIGNIFICANT FOR EBOV REPLICATION. THE GOAL OF THIS PROJECT IS TO DEFINE THE CHARACTERISTICS AND MECHANISTIC SIGNIFICANCE OF THESE TWO VP24 BINDING MODES BY EXAMINING REPRESENTATIVE HOST PROTEINS IDENTIFIED IN OUR AP- MS STUDIES, WITH THE HOST PROTEIN ANP32A REPRESENTING IMPA-DEPENDENT VP24 INTERACTORS AND EMD AND ATP1A1 REPRESENTING IMPA-INDEPENDENT VP24 INTERACTORS. IN AIM 1, WE WILL USE STRUCTURAL AND MUTATIONAL APPROACHES TO CHARACTERIZE CRUCIAL BINDING DETERMINANTS THAT MEDIATE IMPA-DEPENDENT AND INDEPENDENT INTERACTIONS. IN AIM 2, WE WILL EXAMINE THE FUNCTIONAL SIGNIFICANCE OF THESE BINDING MODES. USING OUR TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLE (TRVLP) EBOV LIFE CYCLE MODELING SYSTEM, WE WILL EXAMINE THE IMPACT OF THESE HOST PROTEINS ON DIFFERENT STAGES OF THE EBOV LIFE CYCLE. WE WILL ALSO USE THE PREVIOUSLY DESCRIBED EBOLAVP30-EGFP VIRUS TO EXAMINE HOW THESE HOST PROTEINS IMPACT VIRAL REPLICATION. THE RESULTS FROM THESE STUDIES WILL PROVIDE ADDITIONAL CLARITY ABOUT THE IMPORTANCE OF THE VP24-IMPA INTERACTION AND PROVIDE NOVEL INSIGHT INTO CELLULAR PROCESSES TARGETED BY VP24. TOGETHER, THESE FINDINGS WILL ADVANCE OUR UNDERSTANDING OF THE CRUCIAL ROLE FOR VP24 IN EBOV REPLICATION. THE STUDIES PROPOSED HERE WILL ALSO HELP FURTHER MY PERSONAL CAREER GOALS BY PROVIDING NEW TRAINING IN FILOVIRUSES, NUCLEAR TRAFFICKING, STRUCTURAL BIOLOGY AND NEW MEANS TO EXAMINE VIRUS-HOST INTERACTIONS.