PROJECT SUMMARY A significant fraction of the eukaryotic proteome is composed of integral membrane proteins, most of which are inserted and assembled at the endoplasmic reticulum (ER). The diverse biophysical characteristics, topology, posttranslational modifications, and activities of these membrane proteins necessitate distinct cellular pathways and numerous components to ensure their proper biogenesis and function. The goal of this project is to define a new mechanistic role of the zinc metalloprotease ZMPSTE24 in membrane protein biology. ZMPSTE24 has long been a research focus in my laboratory and is important for human health and longevity through its established role in the proteolytic processing of farnesylated prelamin A, precursor of the nuclear scaffold protein lamin A. Defects in this processing step lead to premature aging (progeria) diseases. However, an intriguing new function for ZMPSTE24 in viral defense was recently discovered by others and surprisingly does not require its catalytic activity: ZMPSTE24 confers potent antiviral activity against many enveloped viruses through its interaction with a class of small membrane proteins called interferon-induced transmembrane proteins (IFITM1, 2, and 3). The IFITMs block virus-host cell fusion by a mechanism that involves “rigidifying” host cell membranes. As is the case for IFITMs, the overexpression of ZMPSTE24 robustly protects cells from infection by enveloped viruses, and its proteolytic activity is not needed in this role. Furthermore, depletion of ZMPSTE24 in cells and mice cause them to succumb to viral infection. These findings place ZMPSTE24 at an important position in the cell’s first line of defense against viral infection, likely through a general cell biological role. Here we hypothesize that ZMPSTE24 defines a central component in a known or new pathway for membrane protein biogenesis (insertion, topology, stability/quality control, posttranslational modification, or oligomerization), with IFITM3 as its substrate. Alternatively, ZMPSTE24 may facilitate IFITM3’s membrane rigidifying function in some other way, directly by recruitment to IFITM3, or indirectly by altering the composition or properties of the lipid bilayer. This project represents an exciting new direction in my laboratory’s long-term studies of ZMPSTE24, inspired by the convergence of ZMPSTE24’s newly discovered role in viral defense and the COVID-19 pandemic. Nevertheless, the studies we propose also relate to earlier research in my laboratory on membrane protein topology, trafficking, and ER quality control. Deciphering the antiviral role of ZMPSTE24 via the IFITMs presents an intriguing puzzle that we are primed to solve. We expect the studies proposed here will uncover a new fundamental role(s) for ZMPSTE24 in membrane protein biogenesis or membrane lipid composition or fluidity. Furthermore, insight into the mechanism whereby ZMPSTE24 enables IFITMs to block virus-host-cell fusion could ultimately be ...