For a virus to infect a cell and subsequently cause disease it must first bind to its cellular receptor. The Oldstone laboratory isolated and characterized the cellular receptor, alpha-dystroglycan (alpha-DG) for several arenaviruses including Lassa fever virus (LFV) and LCMV CI 13. We provided evidence that a-DG is the receptor required for infection as: 1) LFV and LFV glycoprotein (GP) bind at high affinity to purified alpha-DG immobilized on membranes; 2) normally permissive cells bearing a null mutation of the DG gene are resistant to LFV infection; 3) reconstitution of alpha-DG expression in null mutant cells using an adenovirus vector restored susceptibility to LFV. We have redesigned a high-output assay to analyze LFVGP-mediated infection of target cells under BSL2 conditions. In the Boger laboratory solution-phase synthetic techniques have been utilized to create unique combinatorial libraries of small molecules that can be screened to identify therapeutic compounds that promote protein-protein interactions (agonists) or inhibit protein-protein interactions (antagonists). We propose three specific aims: First, to screen combinatorial chemical libraries to discover small molecules that inhibit LFVGP-mediated infection of cells using retroviral vectors that contain LFVGP in their envelope. Second, to develop a drug that would neutralize free LFV by engineering anti-viral receptor-bodies, in which the Fab part of an IgG molecule is replaced by a virus-binding alpha-DG fragment. Third, to validate this inhibition in vivo by blocking LCMV CI 13 infection in a mouse model (CI 13 and LFV binds at high affinity to alpha-DG and predictably utilizes the same binding site) with final verification to be done at CDC using the experimentally defined chemical inhibitor(s) and virulent LFV. The initial grant reviewed by the Study Section noted the proposal to be highly significant, exciting and innovative. However, a weakness of a lack of preliminary data was noted. To correct this deficiency we have 1) altered our high-throughput approach to use a luciferase reporter system, 2) provide data to show we can inhibit virus binding without inhibition of laminin binding, 3) provide evidence of construction of antiviral receptor bodies.
