This proposal describes a five-year mentored research career development project focused on the in vivo efficacy of and mechanisms underlying a novel restriction factor, retroCHMP3. Host restriction factors evolve to protect genomes from threats from viruses and transposable elements. The cellular Endosomal Sorting Complexes Required for Transport (ESCRT) pathway is targeted by many viruses for budding and is implicated in retrotransposition, yet is essential for host membrane remodeling, including during cytokinesis. Host evolution to restrict viruses or retrotransposons must therefore preserve key cellular functions, with little room for error. retroCHMP3 is a retrotransposed, truncated and mutated copy of the essential ESCRT gene chmp3 that dominantly inhibits virus budding and LINE-1 retrotransposition in cells. Remarkably, retroCHMP3 has been detoxified so that it does not block cytokinesis. retrochmp3 arose and evolved independently in mice and new world monkeys, suggesting that it confers selective advantages. This proposal tests the hypothesis that retroCHMP3 is a broad-acting restriction factor that evolved for mammalian defense. The experiments in Aim 1 will elucidate 1) the in vivo efficacy of retroCHMP3 against LINE-1 retrotransposition with a mouse model that reports new LINE-1 insertions in the presence or absence of retroCHMP3, and 2) the mechanism by which LINE- 1 activity requires the ESCRT pathway with a combination of cell culture and biochemical assays.
Aim 2 describes the generation of a knock-in mouse which will reversibly and inducibly express retroCHMP3 and will be used to test 1) the extent of L1 retrotransposition restriction in vivo, 2) the in vivo efficacy of retroCHMP3 against viral infection, and 3) the degree of toxicity from in vivo expression of retroCHMP3. The two most important impacts are: 1) understanding the functionality of a new viral and retrotransposition restriction factor in vivo has implications for evolutionary biology, development of anti-viral interventions, and understanding the balance between host cell toxicity and pathogen restriction, and 2) the role of the ESCRT pathway in LINE-1 biology may reveal new functions for the ESCRT pathway, since known ESCRT functions have been shown to be non-essential for LINE-1 retrotransposition. The investigator is a postdoctoral researcher at the University of Utah and has enlisted two strong mentors with expertise in the ESCRT pathway, virus budding, evolutionary biology, and postdoc mentoring. The candidate is a veterinary scientist who proposes to use this mentored training period to augment her expertise in infectious disease pathogenesis with virology, host-pathogen coevolution, and mechanistic cell biology. The scientific proposal is complemented by a thorough training plan which emphasizes proposal writing, idea generation, management and leadership, in addition to the development of research skills in a new field. The short-term goals and specific training opportunities described in the training plan contribute to the candidate?s long-term goal of leading an independent research program in infectious disease.
This career development award supports the transition to independence for a veterinary scientist with an interest in infectious disease. In this application, our goal is to investigate 1) the effectiveness of a new anti- viral and genome-protective protein in animal models and 2) the cell biology of selfish DNA sequences that litter our genomes. The findings will contribute to the basic understanding of host-pathogen conflicts and cellular biology.
