Project Grant F31AI203115

DEFINING THE REGIONS OF THE ARENAVIRUS MATRIX PROTEIN THAT DRIVE VIRAL BUDDING - PROJECT SUMMARY ARENAVIRUSES ARE A FAMILY OF RODENT-BORNE ENVELOPED RNA VIRUSES THAT CAN CAUSE SEVERE DISEASE FOLLOWING ZOONOTIC SPILLOVER INTO HUMANS. PRESENTLY, THERE ARE NO FDA-APPROVED TREATMENTS OR VACCINES, WHICH REPRESENTS A COMPLETELY UNMET MEDICAL NEED. MOLECULARLY THESE VIRUSES ARE SIMPLE WITH ONLY 4 ENCODED PROTEINS THAT CAN REWIRE HOST CELL PROCESSES. THE VIRAL MATRIX PROTEIN (Z) IS THE SMALLEST OF THE 4 PROTEINS AND IS HIGHLY MULTIFUNCTIONAL. THIS PROTEIN DRIVES THE ASSEMBLY AND BUDDING PROCESS, ANTAGONIZES HOST TYPE I IFN RESPONSES, AND INHIBITS THE VIRAL RNA-DEPENDENT RNA POLYMERASE. WHILE MUCH WORK HAS BEEN DONE TO UNDERSTAND THESE KNOWN Z FUNCTIONS, LITTLE PROGRESS HAS BEEN MADE TO MAP THE KEY REGIONS OF Z (I.E. INDIVIDUAL AMINO ACID RESIDUES OR MOTIFS) THAT DRIVE THESE VARIOUS FUNCTIONS. THIS GRANT WILL FOCUS ON Z'S ABILITY TO DRIVE THE VIRAL BUDDING PROCESS FOR TWO CLASSES OF VIRAL PARTICLES, STANDARD INFECTIOUS PARTICLES AND DEFECTIVE INTERFERING PARTICLES (DIPS). IT IS KNOWN THAT THE PRODUCTION OF DIPS REQUIRES A Z-ENCODED LATE DOMAIN AND RECRUITMENT OF THE CELLULAR ESCRT PATHWAY, WHILE INFECTIOUS PARTICLES DO NOT. THE SPECIFIC Z RESIDUES THAT CONTROL THE RELEASE OF INFECTIOUS PARTICLES, AND THOSE THAT CONTROL THE SWITCH BETWEEN INFECTIOUS AND DEFECTIVE INTERFERING PARTICLES PRODUCTION REMAIN UNKNOWN. TO FILL THIS KNOWLEDGE GAP, I HAVE GENERATED INDIVIDUAL RLCMV VIRUSES THAT ENCODE A SINGLE ALANINE POINT MUTATION ACROSS THE ENTIRE CODING REGION OF THE Z PROTEIN. THROUGH ANALYSIS OF MULTI-CYCLE GROWTH CURVES OF THESE VIRUSES IN HUMAN CELLS IN VITRO, 30 OF THE SCREENED MUTATIONS RESULTED IN ATTENUATED VIRAL PROPAGATION COMPARED TO WILD TYPE LCMV. THIS GRANT AIMS TO USE THESE LOSS-OF-FUNCTION RLCMV Z-ALA MUTANT VIRUSES TO CHARACTERIZE INDIVIDUAL FUNCTIONAL RESIDUES OR MOTIFS THAT ARE REQUIRED TO PRODUCE INFECTIOUS PARTICLES AND/OR DEFECTIVE INTERFERING PARTICLES. IN ADDITION TO CHARACTERIZING THESE FUNCTIONAL RESIDUES IN HUMAN CELLS, AIM 1 OF THIS GRANT WILL ASSESS IF THE BUDDING OF VIRIONS IS CONTROLLED BY THE SAME RESIDUES OR MOTIFS IN RESERVOIR RODENT CELLS. AIM 2 WILL ASSESS IF THE MOST POTENT LOSS-OF-FUNCTION RLCMV Z-ALA MUTANTS ARE ATTENUATED IN RODENT MODELS OF INFECTION AS A MEANS OF PROVIDING PROOF-OF-CONCEPT FOR WHETHER TARGETING THESE RESIDUES COULD BE A METHOD TO GENERATE LIVE-ATTENUATED VACCINES FOR ARENAVIRUSES. THIS PROPOSAL WILL BE AN IDEAL TRAINING FELLOWSHIP IN BOTH CELLULAR AND MOLECULAR VIROLOGY, AND ANIMAL HANDLING AND USE UNDER HIGH BIOSAFETY CONTAINMENT CONDITIONS AT THE UVM BSL3 FACILITY. THIS PROPOSED PLAN WILL AFFORD ME THE OPPORTUNITY TO ENHANCE MY TRAINING IN THE BOTTEN LAB'S BSL2 AND BSL3 LABORATORIES, AND DEVELOP SKILLS IN PROJECT MANAGEMENT, SCIENTIFIC COMMUNICATION, ENTREPRENEURSHIP, AND LEADERSHIP. BROADLY, THIS PROPOSAL WILL MAP THE FUNCTIONAL LANDSCAPE OF THE LCMV Z PROTEIN, DEFINE THE REGIONS OF Z THAT DRIVE VIRAL BUDDING, AND DETERMINE IF ANY SELECTED LOSS-OF- FUNCTION MUTATIONS LEAD TO IN VIVO ATTENUATION WHILE REMAINING IMMUNOGENIC. FUNCTIONALLY IMPORTANT REGIONS OF Z MAPPED THROUGH THESE STUDIES THAT ARE NOT INVOLVED IN VIRAL BUDDING WILL SERVE AS A ROADMAP FOR THE FIELD TO UNDERSTAND HOW Z MEDIATES OTHER KNOWN FUNCTIONS AND POTENTIALLY DEFINE YET-TO-BE DISCOVERED FUNCTIONS.