Enveloped viruses, which are bounded by their own lipid bilayer membranes, require fusion with host cell membranes for transmission and infection. As enveloped human viruses, such as influenza, HIV, and Zika, are responsible for billions of infections and millions of deaths annually, an increasingly acute need for new and novel antiviral therapeutics exists. In analogy to antibiotics, so-called ?broad spectrum? antivirals, effective against many different enveloped viruses, could be particularly efficacious. One potential source of broad spectrum antivirals is the innate immune system of the host organism. A varied repertoire of antiviral proteins, canonically referred to as ?viral restriction factors,? is produced by interferon stimulation of the host cell in response to the presence of viral pathogens. In 2009, a family of small (~15kD) single transmembrane-helix integral membrane proteins, the Interferon-Induced Transmembrane (IFITM) proteins, were identified as host- response viral restriction factors that mediated cellular resistance to infection by Influenza, West Nile and Dengue (enveloped) viruses. These observations have subsequently been extended to approximately twenty different enveloped viruses. Subsequent research, published in 2017, indicated the integral membrane protein zinc metalloprotease ZMPSTE24 as a downstream effector of the human IFITM3 protein. Strikingly, this work indicated that neither the IFITM3 protein nor ZMPSTE24 proteolytic activity is necessary for antiviral behavior; specifically, ?catalytically-dead? ZMPSTE24 appears to be both necessary and sufficient to reduce infection by flu (and, in that publication, by six other enveloped viruses) as determined via cell-based assays. During the last seven years, we have been deeply engaged with understanding the function and structure of ZMPSTE24, initiated by determination of the x-ray crystal structure of a fungal ortholog, and followed by structural and enzymological studies of the human ortholog ZMPSTE24. Preliminary results from classical bulk/ensemble membrane fusion assays show that the presence of ZMPSTE24 significantly inhibits fusion by influenza virus. Both bulk/ensemble membrane fusion assays and Total Internal Reflection Fluorescence (TIRF) ?single-event? microscopy show that the presence of ZMPSTE24 significantly reduces fusion by Ebola virus GP2 fusion protein (EbovGP2). ZMPSTE24 appears to cause no significant reduction in hemifusion (i.e., lipid mixing of fusogen and target membranes), thus seeming to inhibit the specific step of fusion pore formation. Additional preliminary experiments suggest both a direct physical interaction between EbovGP2 and ZMPSTE24, and a reduction in total binding of EbovGP2 to membranes containing ZMPSTE24 vs. those absent of it. Lastly, we have expressed and purified human IFITM3 for inclusion, with ZMPSTE24, in functional and structural studies. We propose to use multiple functional, biochemical, biophysical, and structural techniques to elucidate how an (enzymatically- inactive) membrane-bound protease is able to confer broad spectrum viral resistance.
Viruses such as influenza, Zika and HIV, which are responsible for billions of infections and millions of deaths annually, are transmitted and cause infection by fusing their viral membranes with the cellular membranes of the target organism. In 2017, ZMPSTE24, a protein located in the membranes of humans, was discovered to be highly effective at preventing infection by a remarkably wide range of different viruses. Using a variety of functional and structural approaches, we are seeking to understand, on a molecular level, how ZMPSTE24 is able to provide this resistance to viral infection, and whether ZMPSTE24 could potentially be utilized as an antiviral therapy.
