Project Grant F31AI197582

ELUCIDATING SPEN-DRIVEN M6A REGULATION DURING HENIPAVIRUS INFECTION - PROJECT SUMMARY HENIPAVIRUSES ARE HIGHLY LETHAL, BAT-BORNE BSL-4 RNA VIRUSES THAT HAVE BEEN DECLARED PRIORITY PATHOGENS BY THE WORLD HEALTH ORGANIZATION DUE TO THEIR HIGH CASE FATALITY RATES, LACK OF ANTIVIRALS, AND GLOBAL EMERGENCE. HOWEVER, RESEARCH ON HENIPAVIRUSES IS HINDERED BY STRICT BIOCONTAINMENT REQUIREMENTS AND A LACK OF TRACTABLE MODELS, LEAVING CRITICAL ASPECTS OF INFECTION LARGELY UNEXPLORED. THIS RESEARCH AIMS TO ELUCIDATE HOW HENIPAVIRUSES EXPLOIT CELLULAR PROTEINS, SPECIFICALLY SPEN FAMILY PROTEINS, TO REMODEL VIRAL M6A LANDSCAPES AND HIJACK REGULATORY PATHWAYS TO ULTIMATELY DRIVE INFECTION. THROUGH THE NOVEL USE OF CEDAR VIRUS (CEDV), A BSL-2 HENIPAVIRUS SURROGATE, COUPLED WITH VIRAL CROSSLINKING AND SOLID-PHASE PURIFICATION (VIR- CLASP), I HAVE IDENTIFIED 146 HUMAN PROTEINS DIRECTLY BOUND TO THE PRE-REPLICATED CEDV GENOME, ESTABLISHING THE FIRST HENIPAVIRUS RNA INTERACTOME. AMONG THESE, I UNCOVERED SPEN FAMILY PROTEINS, SPEN, RBM15, AND RBM15B, AS KEY HOST FACTORS THAT ACTIVELY PROMOTE VIRAL INFECTION. USING DIRECT RNA SEQUENCING, I DETERMINED THAT SPEN DEPLETION INDUCES WIDESPREAD HYPOMETHYLATION, AFFECTING ~98% OF DIFFERENTIALLY MODIFIED M6A SITES, ~87% OF WHICH LOCALIZE TO THE L MRNA TRANSCRIPT ENCODING THE VIRAL RNA-DEPENDENT RNA POLYMERASE. GIVEN MY CURRENT FINDINGS, I HYPOTHESIZE THAT RBM15/15B MODULATES M6A METHYLATION DEPOSITION, AND THAT SPEN FAMILY PROTEINS ARE FUNCTIONALLY CONSEQUENTIAL ON VIRAL TRANSCRIPT STABILITY AND/OR TRANSLATION. I WILL TEST THIS HYPOTHESIS IN TWO SPECIFIC AIMS: IN AIM 1, I WILL PROFILE RBM15/15B-DRIVEN M6A REGULATION BY USING NANOPORE DIRECT RNA SEQUENCING TO IDENTIFY DIFFERENTIALLY MODIFIED M6A SITES IN CEDV MRNA TRANSCRIPTS. IN AIM 2, I WILL EVALUATE SPEN-DEPENDENT RIBOSOME OCCUPANCY AND TRANSCRIPT STABILITY OF CEDV MRNAS USING HIGH-THROUGHPUT SEQUENCING APPROACHES. COLLECTIVELY, MY PROPOSAL WILL ELUCIDATE THE UNEXPLORED MOLECULAR MECHANISMS OF HENIPAVIRUS PATHOGENESIS, SPOTLIGHT A PREVIOUSLY UNAPPRECIATED ROLE FOR SPEN FAMILY PROTEINS IN DRIVING VIRAL INFECTION, AND UNCOVER POTENTIAL CELLULAR TARGETS TO AID THE DEVELOPMENT OF HOST-DIRECTED ANTIVIRALS.