ICAM-1 RECEPTOR ANALOGUES AS ANTIRHINOVIRUS AGENTS

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The major group of human rhinovirus (HRV) are responsible for 36 to 45% of all common colds, tens of millions of lost school and work days, billions of dollars of doctor's office visits and over-the-counter remedies, secondary bacterial infections, and exacerbations of respiratory diseases. We will use the cellular receptor for the major group of rhinoviruses, ICAM-l, to develop novel chimeric ICAM-l analogues with improved anti-viral activity. Furthermore, we will study the 3-dimensional structure of the binding site on ICAM- l for rhinovirus, and the mechanism of neutralization and disruption of rhinovirus by ICAM-l analogues, so that small drug molecules with anti- viral activity can be developed. We will prepare improved ICAM-l fragments that contain IgSF domains 1 and 2 of ICAM- l and that have the same affinity for rhinovirus as ICAM-l with all 5 IgSF domains, as demonstrated by surface plasmon resonance spectroscopy with BIAcore. Purified fragments expressed in CHO cell lectin-resistant mutants that have homogenous glycosylation will be crystallized directly or after cleavage between domains 2 and 3 with proteases. Several different kinds of crystals have already been obtained and the 3-dimensional structure will be determined in collaboration with Drs. Stephen Harrison and Jia- huai Wang. Longer term goals are to obtain the structure of an ICAM- 1 fragment complexed with rhinovirus and of a longer ICAM- l fragment. We will prepare improved multivalent chimeras that contain ICAM- l IgSF domains 1-5 or 1-2 fused to hinge and/or Fc regions of IgG, IgA, and IgM. These will be tested for inhibition of binding of rhinovirus to cells, plaque reduction, and disruption of HRV. We will test the hypothesis that chimeras will demonstrate differing efficacy in different assays, and that chimeras with improved efficacy will be generated by using fully active 2 domain ICAM- l fragments and including two more subclasses (IgG and IgM) in the analysis of 5 domain ICAM- l chimeras. We will measure the effective affinity of all the chimeras for HRV with BIAcore. Finally, we will measure affinity and number of binding sites for ICAM-l on disrupted virions and natural empty capsids, and examine the mechanism of RNA release and disruption by testing the hypothesis that disruption is not concerted for the virion as a whole, but can occur in a stepwise fashion, possibly pentamer-by-pentamer. This hypothesis will be tested by measuring ICAM-1 binding and disruption in real time in BlAcore, and by measuring binding of conformation-specific mAb to 80S particle preparations that are hypothesized to differ in % of disrupted pentamers.