The Farzan and Choe laboratories have worked closely to identify the obligate cellular receptors for the SARS coronavirus (SARS-CoV) and for a family of New World hemorrhagic fever arenaviruses. Following these initial studies, we have extensively characterized these receptors and their entry proteins, with focus on implications for zoonotic transmission, inhibition of entry, and fusion mechanisms of these viruses. We identified these receptors by: (1) identifying fragments of the viral entry protein that best bound virus permissive, but not non-permissive, cells, (2) confirming the functional role of these fragments by showing that they specifically inhibited viral entry, (3) further modifying these fragments to improve their biochemical properties while retaining or improving there ability to inhibit entry, (4) optimizing detergent and lysis conditions to enhance precipitation of specific proteins from permissive cells, (5) identifying candidate proteins through tandem mass spectrometry, and (6) proving their role as obligate receptors using a variety of established virological techniques. Our experience to date suggests that this approach can be used to identify receptors of additional viruses that are NIAID priority pathogens. Here we seek to refine this basic approach and identify several of these receptors. We will initially focus on members of three classes of entry proteins, each with a distinct set of challenges and technical hurdles. These are (1) the class I fusion proteins of marburg- and ebolavirus, filoviruses which we have demonstrated use a common receptor, (2) the prototypical class II fusion proteins of dengue II and West Nile flaviviruses, which may or may not use a common receptor, and (3) the fusion protein of Rift Valley Fever virus, a bunyavirus whose fusion protein is largely uncharacterized but which shares properties with both class I and class II fusion proteins.
As we have shown with SARS-CoV and several hemorrhagic fever arenaviruses, identification of obligate viral receptors provides as necessary foundation for understanding viral zoonosis and pathogenesis, and for development entry inhibitors and subunit vaccines. We focus here on five NIAID priority pathogens. Our studies will also develop and refine methods for identification of additional viral receptors.
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